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In Vitro Demonstration of Human Lipoyl Synthase Catalytic Activity in the Presence of NFU1

  • Douglas M. Warui
    Douglas M. Warui
    Department of Chemistrythe , The Pennsylvania State University, University Park, Pennsylvania 16802, United States
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    Orcidhttps://orcid.org/0000-0001-8950-9433
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  • Debangsu Sil
    Debangsu Sil
    Department of Chemistrythe , The Pennsylvania State University, University Park, Pennsylvania 16802, United States
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    Orcidhttps://orcid.org/0000-0002-0718-3925
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  • Kyung-Hoon Lee
    Kyung-Hoon Lee
    Department of Chemistrythe , The Pennsylvania State University, University Park, Pennsylvania 16802, United States
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  • Syam Sundar Neti
    Syam Sundar Neti
    Department of Chemistrythe , The Pennsylvania State University, University Park, Pennsylvania 16802, United States
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  • Olga A. Esakova
    Olga A. Esakova
    Department of Chemistrythe , The Pennsylvania State University, University Park, Pennsylvania 16802, United States
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    Orcidhttps://orcid.org/0000-0001-8377-8368
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  • Hayley L. Knox
    Hayley L. Knox
    Department of Chemistry and Biochemistry and Molecular Biology and the Howard Hughes Medical Institute, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
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  • Carsten Krebs*
    Carsten Krebs
    Department of Chemistry  and  Biochemistry and Molecular Biologythe , The Pennsylvania State University, University Park, Pennsylvania 16802, United States
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-3302-7053
  • , and 
  • Squire J. Booker*
    Squire J. Booker
    Department of Chemistry,  Biochemistry and Molecular Biology  and  Howard Hughes Medical Institutethe , The Pennsylvania State University, University Park, Pennsylvania 16802, United States
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-7211-5937
Cite this: ACS Bio Med Chem Au 2022, 2, 5, 456–468
Publication Date (Web):June 13, 2022
https://doi.org/10.1021/acsbiomedchemau.2c00020

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Lipoyl synthase (LS) catalyzes the last step in the biosynthesis of the lipoyl cofactor, which is the attachment of sulfur atoms at C6 and C8 of an n-octanoyllysyl side chain of a lipoyl carrier protein (LCP). The protein is a member of the radical S-adenosylmethionine (SAM) superfamily of enzymes, which use SAM as a precursor to a 5′-deoxyadenosyl 5′-radical (5′-dA·). The role of the 5′-dA· in the LS reaction is to abstract hydrogen atoms from C6 and C8 of the octanoyl moiety of the substrate to initiate subsequent sulfur attachment. All radical SAM enzymes have at least one [4Fe–4S] cluster that is used in the reductive cleavage of SAM to generate the 5′-dA·; however, LSs contain an additional auxiliary [4Fe–4S] cluster from which sulfur atoms are extracted during turnover, leading to degradation of the cluster. Therefore, these enzymes catalyze only 1 turnover in the absence of a system that restores the auxiliary cluster. In Escherichia coli, the auxiliary cluster of LS can be regenerated by the iron–sulfur (Fe–S) cluster carrier protein NfuA as fast as catalysis takes place, and less efficiently by IscU. NFU1 is the human ortholog of E. coli NfuA and has been shown to interact directly with human LS (i.e., LIAS) in yeast two-hybrid analyses. Herein, we show that NFU1 and LIAS form a tight complex in vitro and that NFU1 can efficiently restore the auxiliary cluster of LIAS during turnover. We also show that BOLA3, previously identified as being critical in the biosynthesis of the lipoyl cofactor in humans and Saccharomyces cerevisiae, has no direct effect on Fe–S cluster transfer from NFU1 or GLRX5 to LIAS. Further, we show that ISCA1 and ISCA2 can enhance LIAS turnover, but only slightly.

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Introduction

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Lipoic acid is an eight-carbon straight-chain fatty acid containing sulfur atoms at C6 and C8. (1−3) It is found in all domains of life. (4−11) Its primary cellular function is as a cofactor in several multienzyme complexes that are involved in energy metabolism and the catabolism of certain amino acids. (7,11) In humans, these complexes include the pyruvate dehydrogenase complex (PDC), the α-ketoglutarate dehydrogenase complex (KGC), the branched chain oxo-acid dehydrogenase complex (BCODC), the glycine cleavage system (GCS), and the α-ketoadipate complex (KAC), all of which are found in the mitochondrion. (12,13) In each of these complexes, lipoic acid is tethered covalently in an amide linkage to a target lysyl residue of a lipoyl carrier protein (LCP), producing a 14 Å flexible appendage that can access active sites of other component proteins. Very little free lipoic acid exists in the cell in the absence of supplementation. In fact, the molecule is biosynthesized in its cofactor form rather than as the free acid. This biosynthetic pathway involves a bacterial-type acyl carrier protein (ACP) upon which the C8 fatty acyl backbone is constructed. In humans, LIPT2, an octanoyltransferase, is believed to transfer the octanoyl chain from octanoyl–ACP to the target lysyl residue only of the H protein, the LCP of the GCS. Next, lipoyl synthase (LS, LIAS in humans and LipA in Escherichia coli) attaches thiol groups at C6 first, and then at C8, to give the intact lipoyl cofactor. (13,14) Finally, LIPT1, a lipoyltransferase, is believed to distribute the lipoyl appendage to other LCPs (13,15) (Figure 1). This biosynthetic pathway differs from the canonical pathway in E. coli wherein octanoyltransferase transfers an octanoyl group directly to each of the LCPs, which include the H protein of the GCS and the E2 subunits of the KGC and PDC. (16−21) LipA then acts on each of the octanoyllysyl–LCPs to generate the respective lipoyl cofactor. (22−24)

Figure 1

Figure 1. Proposed de novo biosynthetic scheme of lipoyl cofactor in humans.

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LS belongs to the radical S-adenosylmethionine (SAM) superfamily of enzymes, which use a [4Fe–4S] cluster cofactor to cleave SAM reductively to produce a 5′-deoxyadenosyl 5′-radical (5′-dA·). (24−33) In LS catalysis, the 5′-dA· is used to abstract hydrogen atoms (H·) from C6, and then from C8, of the octanoyllysyl residue to allow for sulfur attachment. (34−37) Unlike most radical SAM (RS) enzymes, which contain only one [4Fe–4S] cluster, LS contains two [4Fe–4S] clusters. (34,38−40) One cluster, termed the RS cluster, is found in all RS enzymes and is ligated by three cysteine residues in a conserved CxxxCxxC motif. SAM binds to the unligated iron ion of this cluster, which is a prerequisite for its reductive cleavage. (27,41−43) The second cluster, termed the auxiliary cluster, is bound by cysteines in an N-terminal CxxxxCxxxxxC motif and a C-terminal serine in a conserved RSSY motif (Figure 2). This cluster has been shown to be the source of the appended sulfur atoms, resulting in its degradation as a result of turnover. (36,44,45) Therefore, LS catalyzes no more than 1 turnover during in vitro reactions in the absence of a system that restores the auxiliary [4Fe–4S] cluster. (46,47)

Figure 2

Figure 2. Sequence alignment of LS proteins from E. coli (P60716), M. tuberculosis (P9WK91), T. elongatus (Q8DLC2), and humans (O43766). The conserved residues that ligate the auxiliary and the RS clusters of LS are highlighted in blue and red, respectively.

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Recently, it was shown that the iron–sulfur (Fe–S) cluster carrier protein E. coli NfuA can efficiently regenerate the auxiliary [4Fe–4S] cluster of E. coli LipA, permitting LipA to perform multiple turnovers. (46,47) Fe–S cluster assembly and repair is a highly regulated process that is coordinated by a complex network of proteins. In bacteria, yeast, and human model systems, de novo biogenesis of Fe–S clusters involves transient assembly of clusters on the scaffold protein ISCU (IscU in bacteria), with their subsequent transfer directly to recipient apo acceptor proteins or to a subset of late-acting carrier proteins. (48−55) Among others, mitochondrial proteins BOLA3, ISCA1, ISCA2, GLRX5, and NFU1 have been implicated in Fe–S cluster assembly and trafficking in humans. (52,56−58) Select mutations in genes encoding these proteins have been reported to be pathogenic and to cause severe infantile disorders of systemic energy metabolism and multiple mitochondrial dysfunctions syndrome types 1–5 (MMDS). (48,59,60) Some of the severe manifestations of MMDS include weakness, respiratory failure, lack of neurologic development, hyperglycemia, lactic acidosis, and early death, (61−64) symptoms associated with defective lipoic acid biosynthesis. Biochemical features of patients with pathogenic mutations in genes encoding these proteins include decreased activities of several mitochondrial Fe–S cluster-containing enzymes, including complex-I, complex-II, and LIAS. (65) These studies suggest that BOLA3, ISCA1, ISCA2, GLRX5, and NFU1 may play important roles in Fe–S cluster biogenesis, trafficking, and regeneration mechanisms, especially for LIAS. However, exactly how these proteins function remains elusive.
To date, our understanding of the mechanism of lipoyl cofactor formation is derived mainly from in vivo and in vitro studies of the E. coli enzyme, (10,24,34,36,37,39,66−69) with additional insight from studies of enzymes from Sulfolobus solfataricus, (70)Thermosynechococcus elongatus, (44) and Mycobacterium tuberculosis. (71,72) Only recently has human LIAS been isolated and investigated in vitro. In one study, LIAS was used to show how paramagnetic NMR can be leveraged to demonstrate SAM and substrate binding to RS enzymes. (73) In a second study, the effect of ISCA2 and ISCU [among other iron sulfur cluster (ISC) assembly proteins] on the de novo in vitro reconstitution of the Fe–S clusters on LIAS was assessed. (74) However, neither study determined LIAS activity quantitatively nor specifically addressed how the auxiliary cluster of LIAS is restored during turnover. The lack of robust in vitro biochemical studies on LIAS, and particularly how the implicated Fe–S cluster trafficking proteins BOLA3, ISCA1, ISCA2, GLRX5, and NFUI function in lipoyl cofactor biosynthesis, led us to investigate how the auxiliary cluster of LIAS is regenerated after turnover.
Herein, we show that NFU1 and LIAS form a tight complex in vitro, as has been shown in in vivo yeast two-hybrid studies, (75) and that NFU1 can efficiently restore the auxiliary cluster of LIAS during turnover. We also investigate several additional Fe–S cluster carrier proteins, including BOLA3, previously identified as being critical in the biosynthesis of the lipoyl cofactor in humans and Saccharomyces cerevisiae. However, our in vitro studies suggest that BOLA3 has no direct effect on Fe–S cluster transfer from NFU1 or GLRX5 to LIAS. Further, we show that ISCA1 and ISCA2 can enhance LIAS turnover, but only slightly.

Results

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Isolation and Characterization of LIAS

Full-length human LIAS (UniProtKB O43766) is composed of 372 amino acids; however, there are several predicted isoforms of shorter length. Importantly, the first 27 amino acids are predicted to form the mitochondrial targeting sequence. When we attempted to express this full-length construct, almost all the protein was produced in inclusion bodies. Similar to two recent publications, (73,74) we therefore engineered a construct (amino acids 28–372) that lacked the mitochondrial targeting sequence, which expressed well in E. coli and was amenable to purification with its two [4Fe–4S] cluster cofactors largely intact. This protein contained an N-terminal SUMO tag, which was removed after purification, affording an LIAS variant containing a Gly-His N-terminal appendage. The LIAS gene was expressed along with genes on plasmid pDB1282, which harbors the Azotobacter vinelandiiisc operon. (76) This coexpression strategy has been shown to enhance the production of soluble E. coli LipA as well as its Fe–S cluster content. (34) The protein was purified under anoxic conditions using Ni–NTA immobilized metal affinity chromatography (Ni–IMAC) followed by size-exclusion chromatography (SEC) to remove impurities such as protein aggregates and unbound iron and sulfide species. Upon purification, the protein was found to be ≥95% homogeneous (Figure 3A) and contained 9.5 ± 0.1 iron and 5.5 ± 0.1 sulfide ions per polypeptide after accounting for a correction factor of 1.6 for the Bradford method of protein quantification, which was established by quantitative amino acid analysis. The UV–vis spectrum (Figure 3B) showed a broad absorption feature at ∼410 nm typical of [4Fe–4S] cluster binding proteins. The 4.2 K/53 mT Mössbauer spectrum of LIAS (Figure 3C) is dominated by a quadrupole doublet with parameters [isomer shift (δ) = 0.46 mm/s, quadrupole splitting (ΔEQ) = 1.14 mm/s; ∼87% of total intensity, 1.7 equiv of [4Fe–4S] per LIAS, blue line] typical of [4Fe–4S]2+ clusters. (77) The shoulder indicated by the arrow is at a position typically observed for the “Fe(II)-like” site of a site-differentiated [4Fe–4S]2+ cluster. (36) Analysis of the field dependence of spectra collected in a 53 mT magnetic field and zero field (Figure S1) suggests the presence of ∼0.3 equiv of [3Fe–4S]0 cluster. The electron paramagnetic resonance (EPR) spectrum of purified LIAS after reduction with dithionite and recorded at 10 K (Figure 3D) shows a rhombic signal typical of [4Fe–4S]+ clusters, with estimated g-values of 2.02, 1.92, and 1.86. The UV–vis, Mössbauer, and EPR spectra in concert with iron and sulfide quantification are consistent with the presence of two [4Fe–4S]2+ clusters, as has been shown previously for human LIAS (74) and the E. coli, (34)M. tuberculosis, (71,72) and T. elongatus enzymes. (44) It should be noted that our iron quantification shows 1.5 irons beyond the eight that our model suggests and that this additional iron is not observed in the Mössbauer spectrum. We believe that the discrepancy is due to systematic inaccuracies in iron and protein quantification.

Figure 3

Figure 3. Biophysical characterization of LIAS: SDS–PAGE gel analysis of the expression and purification of LIAS on a Ni–NTA column (A), UV–vis scan of LIAS (B), Mössbauer spectrum of LIAS (C), and EPR spectrum of dithionite-reduced LIAS (D). (A) Lane 1, pre-IPTG induction; lane 2, post-IPTG induction; lane 3, crude lysate; lane 4, Ni–NTA column flow-through; lane 5, wash; lane 6; SUMO–LIAS fusion eluate; lane 7, LIAS after the SUMO tag is removed; lane 8, protein molecular weight ladder. (B) The UV–vis absorption scan of 8 μM purified LIAS showing a broad absorption at ∼410 nm, which is typical for proteins that bind [4Fe–4S] clusters. (C) The Mössbauer spectrum of 380 μM LIAS at 4.2 K, collected in the presence of a 53 mT external magnetic field applied parallel to the direction of propagation of the γ beam. The vertical bars represent the experimental spectrum, and the blue line shows the features associated with a [4Fe–4S]2+ cluster. The arrow indicates the shoulder resulting from spectral features of a site-differentiated [4Fe–4S]2+ cluster. (D) The EPR spectrum of 400 μM LIAS reduced with 4 mM dithionite and collected at 10 K with 10 mW microwave power and 0.2 mT modulation amplitude confirming bound [4Fe–4S] clusters.

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Isolation and Characterization of NFU1

In a recent in vitro study, E. coli NfuA was shown to regenerate the auxiliary cluster of E. coli LipA after each turnover, thus allowing LipA to act catalytically. (47) There have also been several recent in vitro studies of NFU1, the human ortholog of E. coli NfuA. In one study, focused on NMR solution structures of NFU1, it was shown that holo-NFU1 could donate its [4Fe–4S] cluster to apo aconitase, (78) while in another, holo-NFU1 was reported to transfer a [2Fe–2S] cluster to apo ferredoxin 1 and ferredoxin 2. (79) Given the effects of E. coli NfuA on E. coli LipA turnover and the activation of aconitase by NFU1, we assessed the effect of NFU1 on LIAS turnover. Like NfuA, NFU1 is bimodular, with a degenerate N-terminal A-type domain and a highly conserved NifU-like C-terminal Fe–S cluster binding domain. Two cysteine residues in a conserved C-terminal CxxC motif are believed to serve as ligands to a [4Fe–4S] formed at the interface of two monomers both in NfuA and in NFU1 (Figure 4), although there have been disagreements in the literature concerning whether this form is biochemically relevant or whether a form containing a [2Fe–2S] cluster is more relevant. (64,80)

Figure 4

Figure 4. Amino acid sequence alignment of E. coli NfuA (P63020) and human NFU1 (Q9UMS0) showing the two conserved cysteine residues that ligate the Fe–S cluster highlighted in red.

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To study the effect of NFU1 on LIAS activity, we first expressed and purified the full-length construct (amino acids 1–254) and a construct lacking the mitochondrial transit sequence (amino acids 10–254). Unfortunately, both constructs led to poorly behaved protein products that existed mostly in higher order oligomeric states that were difficult to resolve by SEC. We therefore tried a shorter N-terminally truncated variant (amino acids 59–254). The region that was removed is predicted by AlphaFold (81) (AF-Q9UMS0-F1) to be highly unstructured, while the truncated variant is well-structured and has been reported in previous studies. (78,82) The construct was overproduced in the presence of plasmid pDB1282 and expressed as a fusion with an N-terminal SUMO tag that was removed during purification to yield NFU1 containing a Gly-His N-terminal appendage. The protein was isolated under anoxic conditions using Ni–IMAC followed by SEC and shown to be ≥95% homogeneous by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel analysis (Figure 5A). The UV–vis spectrum of the protein, with a distinctive feature at 410 nm, is consistent with the presence of a [4Fe–4S] cluster (Figure 5B), as has been observed in previous studies. (75) Analysis of 57Fe-labeled NFU1 by Mössbauer spectroscopy (Figure 5C) reveals that the spectrum is dominated by a single quadrupole doublet (δ = 0.48 mm/s, ΔEQ = 1.20 mm/s; ∼97% of total intensity, blue line) typical of [4Fe–4S]2+ clusters. (77) When NFU1 was analyzed by EPR, a very weak signal was observed with and without dithionite reduction (Figure S2), suggesting that the [4Fe–4S] cluster is not easily reduced to the [4Fe–4S]+ state. Iron and sulfide analysis indicated that the isolated protein contained 2.40 ± 0.02 irons and 3.00 ± 0.03 sulfides per polypeptide, consistent with a bridging [4Fe–4S] cluster between two NFU1 monomers.

Figure 5

Figure 5. SDS–PAGE analysis of the expression and purification of NFU1 on a Ni–NTA column (A), UV–vis scan of 15 μM NFU1 (B), and Mössbauer spectra of NFU1 (C). (A) Lane 1, pre-IPTG induction; lane 2, post-IPTG induction; lane 3, crude lysate; lane 4, Ni–NTA column flow-through; lane 5, wash; lane 6, SUMO–NFU1 fusion eluate; lane 7, NFU1 after the SUMO tag is removed; lane 8, protein molecular weight ladder. (B) The UV–vis absorption scan spectrum of 15 μM purified NFU1 showing a broad absorption at ∼410 nm indicative of a bound [4Fe–4S] cluster. (C) The 4.2 K Mössbauer spectra of 860 μM NFU1 in the presence of a 53 mT external magnetic field applied parallel to the direction of propagation of the γ beam. The vertical bars represent the experimental spectra, and the blue line shows the features associated with a [4Fe–4S]2+ cluster.

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NFU1 Binds Tightly to LIAS

Our previous studies indicated that E. coli NfuA binds tightly to E. coli LipA, suggesting that NFU1 might interact similarly with LIAS, as has been determined recently through yeast two-hybrid analysis. (75) To assess whether NFU1 and LIAS form a tight complex in vitro, the holo (Fe–S cluster containing) forms of the two proteins were analyzed separately and together by SEC (Figure 6A). LIAS alone (blue trace) elutes at 62.5 mL, exhibiting an experimentally calculated mass of 46 kDa (theoretical mass, 39.2 kDa) based on the elution profiles of a suite of standards. NFU1 alone (black trace) elutes at 67.7 mL, exhibiting an experimentally calculated mass of 29.8 kDa (theoretical mass, 22 kDa). The sample containing both LIAS and NFU1 (red trace) shows an elution volume of 59.1 mL, corresponding to an experimentally calculated mass of 61.2 kDa, suggestive of a 1:1 heterodimer of LIAS and NFU1 (theoretical mass, 61.2 kDa). To confirm the results obtained by SEC, we subjected fractions from the peaks observed in the LIAS alone, NFU1 alone, and LIAS plus NFU1 traces to SDS–PAGE (Figure 6B). As shown, the peak from the LIAS plus NFU1 sample (lane 3) contains both NFU1 and LIAS. In these experiments, NFU1 migrates as a monomer and interacts with LIAS as a monomer. We further characterized the interactions between NFU1 and LIAS by determining the equilibrium binding dissociation constant (KD) using isothermal titration calorimetry (ITC, Figure 6C). The results from ITC indicate a KD value of 0.7 ± 0.2 μM, confirming the strong interaction.

Figure 6

Figure 6. Size-exclusion gel filtration chromatography elution profiles of holo-LIAS (100 μM, blue), holo-NFU1 (200 μM, black), and a 1:1 mixture of holo-LIAS and holo-NFU1 (100 μM each, red) (A). SDS–PAGE analysis of the chromatographed proteins: lane 1, holo-NFU1 alone; lane 2, holo-LIAS alone; lane 3, a mixture of holo-NFU1 and holo-LIAS, indicating complex formation (B). ITC binding results of LIAS titrated into NFU1, showing entropically driven binding with a dissociation constant (KD) of 0.7 ± 0.2 μM (C).

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In Vitro Determination of LIAS Activity

LIAS in vitro activity was determined in assays using a four amino acid peptide substrate mimic containing an octanoyllysyl residue [Glu-(N6-octanoyl)Lys-Ala-Tyr], a shorter version of the eight amino acid peptide previously used in E. coli LipA (36,47) and M. tuberculosis LipA (72) assays. For reasons that we do not understand, this shorter 4-mer peptide affords more turnovers than the longer 8-mer peptide. As shown in Figure 7A, the protein catalyzes formation of no more than 1 equiv of lipoyl product (blue trace), with no significant accumulation of the 6-thiooctanoyl intermediate (black trace), consistent with previous studies of E. coli LipA that indicate that the auxiliary cluster gets degraded as a function of turnover and that both sulfurs are contributed by the same LIAS polypeptide. (68)

Figure 7

Figure 7. LIAS activity determinations: LIAS (10 μM) activity in the absence of NFU1 (A), in the presence of 200 μM NFU1 (B), in the presence of both 200 μM NFU1 and 5 mM sodium citrate (C), and in the presence of 200 μM NFU1 reconstituted with 34S-labeled sulfide (D). LIAS alone catalyzes about 1 turnover of lipoyl product (blue trace) with the 6-thiooctanoyl intermediate quickly reaching a steady level (black trace) (A). The inclusion of an excess of NFU1 in the LIAS reaction promotes multiple turnovers and generation of more than 5 equiv of lipoyl product (red trace), while the formation and decay of the intermediate mimics that of LIAS alone (gray trace) (B). The inclusion of 5 mM sodium citrate, a divalent metal chelator, does not significantly alter the effect of NFU1 (purple trace) compared to reactions in which citrate is omitted (red trace) (C). In the presence of NFU1 reconstituted with 34S2–, the 32S-labeled lipoyl peptide product is formed first before formation of the mixed 32S–34S (blue trace) and 34S–34S-labeled (red trace) lipoyl peptide products (D). The data in panels C and D suggest direct cluster transfer from NFU1 to LIAS during turnover. Unless otherwise noted, all activity assays included the following at their indicated final concentrations: 350 μM octanoyl peptide substrate, 0.75 mM SAM, and 10 μM SAH nucleosidase. The reactions were carried out at room temperature in a buffer that contained 50 mM HEPES, pH 7.5, and 0.25 M KCl and were initiated with a final concentration of 1 mM dithionite. The respective data shown in panels A–D are averages from assays done in triplicate, and the error bars represent one standard deviation from the mean. The 6-thiooctanoyl intermediate data were fit to an exponential equation that accounts for its formation and decay phases (A and B), while the lipoyl peptide product data were fit to a biphasic double-exponential rate of formation equation, assuming an A → B → C model, as has been previously reported for M. tuberculosis LipA (ref (72)).

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In Vitro Determination of NFU1 Effect on LIAS Activity

After establishing that NFU1 binds tightly to LIAS, we assessed whether NFU1 affects LIAS activity. As shown in Figure 7A, LIAS produces not more than 1 equiv of product in the absence of NFU1. By contrast, in the presence of excess NFU1 (200 μM monomer), 10 μM LIAS catalyzes more than 5 turnovers in a time-dependent manner over 150 min (Figure 7B, red trace). The lack of a clear burst followed by a slower phase of product formation suggests that regeneration of LIAS’s auxiliary cluster is not rate-limiting, similar to what was observed with E. coli LipA and NfuA. (47) The amount of observed lipoyl product is less than what would be expected, given that the NFU1 dimer is in a 10-fold excess. Currently, we attribute the leveling off of activity to aberrant chemistry during destruction of the auxiliary cluster and its subsequent reconstitution. We predict that a more physiological system, especially with respect to the choice of reductant, may enhance the extent of turnover. To confirm that cluster transfer from NFU1 to LIAS is largely direct rather than a result of release of iron and sulfide into solution, the effect of NFU1 on LIAS activity was probed in the presence of 5 mM sodium citrate. Under these conditions, citrate will sequester free iron liberated by NFU1 and prevent it from being used to reconstitute LIAS. The observation that the presence of 5 mM citrate shows no dramatic effect on overall lipoyl product formation (Figure 7C) suggests that the transfer is direct, consistent with the finding that NFU1 and LIAS form a complex. The somewhat reduced activity is attributed to the ability of citrate to remove the cluster from NFU1, as was observed previously in experiments conducted with E. coli IscU. (47) Direct cluster transfer was further assessed by performing LIAS activity assays in the presence of an NFU1 protein that was chemically reconstituted with 34S2– to yield NFU1 containing a [4Fe–434S] cluster. Results from these studies show relatively rapid formation of 32S–32S-containing lipoyl product, which is followed by slower production of 32S–34S-containing and 34S–34S-containing lipoyl product (Figure 7D). The formation of lipoyl product containing the mixed isotope (32S–34S) was also observed with the E. coli NfuA/LipA system, although larger amounts of this product are formed with NFU1/LIAS. (47) It is not clear whether mixed-isotope formation is due to the conditions of the assay (e.g., presence of dithionite or incomplete LIAS reconstitution) or is intrinsic to how the cluster is transferred from NFU1/NfuA to LIAS/LipA. In reactions containing NfuA and LipA, almost 2 equiv of 32S–32S-containing lipoyl product was formed before formation of the 34S-containing species. In the NFU1/LIAS reactions herein, it appears that almost 1.5 equiv of the 32S–32S-containing lipoyl product is formed during the reaction. These data suggest that potentially all four sulfides of the auxiliary cluster of LIAS can be used for lipoyl product formation. It should be noted that the total amount of lipoyl product generated when using 34S-reconstituted NFU1 is lower than that when using NFU1 at natural abundance. We believe the difference is related to how each protein is produced. To generate 34S-reconstituted NFU1, the protein is first isolated in its apo form before being reconstituted. By contrast, NFU1 at natural abundance is produced directly in its Fe–S cluster form, and then further reconstituted.

In Vitro Effect of NFU1 plus BOLA3 on LIAS Activity

Previous studies have implicated BOLA3 in lipoyl cofactor formation; however, it has not been established when exactly BOLA3 functions. BOLA3 is a mitochondrial Fe–S cluster assembly and trafficking factor that facilitates Fe–S cluster insertion into a subset of Fe–S cluster acceptor proteins, and it has been suggested that it might act synergistically with NFU1. (83) BOLA3 was previously reported to form heterodimeric complexes with NFU1 and also GLRX5, and its deletion was associated with defective lipoic acid biosynthesis in vivo, suggesting its requirement for LIAS Fe–S cluster maturation. (65,83) With this precedent in mind, we tested whether BOLA3 could act synergistically with NFU1 for Fe–S cluster transfer to LIAS during turnover. However, inclusion of BOLA3 in our LIAS activity assays in the presence of NFU1 does not show any additional effect on turnover (Table 1 and Figure S3A). Moreover, SEC experiments similar to those conducted with LIAS and NFU1 show that BOLA3 does not bind tightly to LIAS.
Table 1. Effect of Fe–S Cluster Assembly Proteins on LIAS Activitya
NFU1BOLA3ISCA1ISCA2ISCUGLRX5effect on LIAS activityb
X     increase to 5–6 turnovers
XX    same as NFU1 alone
  X   increase to ∼1.5 turnovers
   X  increase to ∼2 turnovers
    X slight increase to ∼1 turnover
     Xno observed effect
 X   Xno observed effect
a

X denotes the presence of that protein in reaction mixtures.

b

Due to differences in LIAS cluster content from batch to batch, the observed turnover numbers vary by about 10–20%. We present the best turnover numbers observed for each corresponding protein or protein mixture.

In Vitro LIAS Activity Determination in the Presence of GLRX5, ISCA1, ISCA2, and ISCU

ISCA2 and ISCU have also been reported to be able to reconstitute LIAS, transforming an inactive protein into one that is competent for catalysis. (74) In other work, E. coli IscU was reported to reconstitute E. coli LipA, allowing for multiple turnovers, (47) while in another study, ISCA1, ISCA2, and IBA57 have been implicated in late-stage [4Fe–4S] cluster maturation, including Fe–S clusters in LIAS. (84) On the basis of these studies, we explored the effect of including purified and appropriately reconstituted GLRX5, ISCA1, ISCA2, and ISCU proteins in LIAS activity assays. Additionally, the effect of BOLA3 in activity assays that also included GLRX5 was tested. In summary, when either ISCA1 or ISCA2 are included in LIAS activity assays, only a modest increase in turnover is observed, up to an additional ∼0.5 and ∼1 equiv for ISCA1 and ISCA2, respectively. No change in LIAS turnover is observed by inclusion of ISCU, GLRX5, or BOLA3 in the presence of GLRX5 (Table 1 and Figure S3, parts B and C).

Discussion

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LS reductively cleaves SAM to generate 5′-dA· that abstracts substrate hydrogen atoms to allow for sequential sulfur insertions to produce the lipoyl cofactor. During turnover, LS sacrifices its auxiliary [4Fe–4S] cluster as the source of the sulfur atoms incorporated into the product, a process that leads to degradation of the auxiliary cluster. Thus, the enzyme is rendered inactive after a single turnover in the absence of a system that can regenerate the auxiliary cluster either by repairing it or by fully replacing it. Fe–S clusters are essential in all domains of life and are involved in various cellular processes, including respiration, ribosome assembly, DNA repair, and the biosynthesis of key metabolites. (85−88) Due to the complexity of ISC assembly, trafficking, and repair mechanisms in vivo, these processes are yet to be fully understood. Understanding these mechanisms in humans is vital because Fe–S clusters and their incorporation into proteins that require them are critical to many serious diseases. (55,56,63,89−91) While recent in vitro attempts to characterize Fe–S cluster assembly and trafficking have been reported, the determination of which late-acting carriers mediate LIAS auxiliary cluster regeneration during catalytic turnover remains unresolved. Here, we have investigated the effects of select human mitochondrial Fe–S cluster carriers (BOLA3, ISCA1, ISCA2, ISCU, GLRX5, and NFU1) on LIAS auxiliary cluster reconstitution during turnover and demonstrate for the first time that, like its E. coli counterpart NfuA, human NFU1 can efficiently reconstitute human LIAS during turnover to promote catalytic activity. Further, we show that NFU1 interacts tightly with LIAS, corroborating a similar finding from previously reported yeast two-hybrid experiments. (75) By contrast, ISCA1 and ISCA2 only partially enhance turnover of LIAS. It is likely that ISCA1 and ISCA2 are involved in de novo Fe–S incorporation into LIAS and not incorporation during catalytic turnover. Moreover, our studies suggest that neither BOLA3, GLRX5, nor ISCU are involved in the immediate regeneration of the auxiliary cluster of LIAS during turnover, although it appears that ISCU, like ISCA1 and ISCA2, can reconstitute LIAS de novo. Our studies suggest that the mechanism by which NFU1 transfers its cluster to LIAS during turnover is distinct from de novo reconstitution of LIAS and provide the basis for an additional system on which to investigate this transfer.

Materials and Methods

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Materials

N-(2-Hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid) (HEPES) was purchased from Fisher Scientific. Imidazole was purchased from J. T. Baker Chemical Co. Potassium chloride and glycerol were purchased from EMD Chemicals. 2-Mercaptoethanol (BME), sodium dithionite, phenylmethylsulfonyl fluoride (PMSF), pyridoxal 5′-phosphate (PLP), and sodium sulfide were purchased from MilliporeSigma. Dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), kanamycin, ampicillin, arabinose, and isopropyl β-d-1-thiogalactopyranoside (IPTG) were purchased from Gold Biotechnology. Nickel-nitrilotriacetic acid (Ni-NTA) resin was acquired from Qiagen. S-Adenosyl-l-methionine (SAM) was synthesized and purified as described previously. (92) Restriction enzymes and materials for cloning were obtained from New England Biolabs (Ipswich, MA). DNA isolation kits were purchased from Macherey-Nagel (Dueren, Germany). All other chemicals and materials were of the highest grade available and were from MilliporeSigma.
All peptides used in this study were custom-synthesized by Proimmune (Oxford, U.K.) except the octanoyl-containing peptide substrate [Glu-(N6-octanoyl)Lys-Ala-Tyr], which was synthesized by Genscript (Piscataway, NJ, U.S.A.). AtsA peptide (Pro-Met-Ser-Ala-Pro-Ala-Arg-Ser-Met) was used as an external standard for quantification of peptide products during liquid chromatography–mass spectrometry (LC–MS) analysis. An 8-thiooctanoyl-containing peptide [Glu-(N6-8-thiooctanoyl)Lys-Ala-Tyr] and a lipoyl-containing peptide [Glu-(N6-lipoyl)Lys-Ala-Tyr] were used as product standards for the 6-thiooctanoyl intermediate and lipoyl products, respectively.

General Methods

UV–vis spectra were recorded on a Varian Cary 50 spectrometer (Walnut Creek, CA) using the WinUV software package to control the instrument. Ultraperformance liquid chromatography (UPLC) was conducted on an Agilent Technologies 1290 Infinity II system coupled to an Agilent Technologies 6470 QQQ mass spectrometer (Santa Clara, CA) with detection by tandem mass spectrometry (MS/MS). Data collection and analysis were performed using the associated MassHunter software package.

Plasmids and Strains

Genes encoding Homo sapiens BOLA3 (aa 27–107), ISCA1 (aa 13–129), ISCA2 (aa 9–154), ISCU (aa 35–167), GLRX5 (aa 32–157), LIAS (aa 28–372), and NFU1 (aa 59–254) without their respective mitochondrial targeting sequences were synthesized at ThermoFisher Scientific after codon optimization using GeneArt software (ThermoFisher Scientific) for protein overexpression in E. coli. The genes were subcloned into a modified pSUMO plasmid (LifeSensors Inc.), (pDWSUMO), and pET28a vectors using NdeI and either XhoI or BamHI restriction sites. After sequence verification by DNA sequencing at the Penn State Genomics Core Facility (University Park, PA), the resulting plasmids, pDWSUMO-BOLA3, pDWSUMO-ISCA1, pDWSUMO-ISCA2, pDWSUMO-ISCU, pDWSUMO-GLRX5, pDWSUMO-LIAS, and pDWSUMO-NFUI (and their corresponding pET28a counterparts), were separately used to transform E. coli BL21 (DE3) competent cells containing the pDB1282 plasmid, which harbors the isc operon from A. vinelandii. (93) As cloned, each of the proteins in the pDWSUMO plasmid was expressed as a fusion with an N-terminal SUMO tag that also contained a His6-tag at the N-terminus, while their counterparts cloned in pET28a were expressed with an N-terminal His6-tag. During purification, the SUMO tag was removed using ULP1 protease, affording corresponding pure proteins with a Gly-His appendage at the N-terminus.

Growth and Expression of BOLA3, ISCA1, ISCA2, ISCU, GLRX5, LIAS, and NFU1

All proteins were overexpressed in E. coli using the following general procedure with minor adjustments for BOLA3 since it is not an Fe–S carrier protein by itself. In a typical growth, a 200 mL starter culture containing 25 μg/mL kanamycin (50 μg/mL for BOLA3) and 50 μg/mL ampicillin (no ampicillin was included for BOLA3) was inoculated with a single colony and incubated overnight at 37 °C with shaking at 250 rpm. A 25 mL aliquot of the starter culture was used to inoculate 4 L of M9 minimal medium containing appropriate antibiotic(s) as above and incubated at 37 °C with shaking (180 rpm) until an optical density at 600 nm (OD600) of 0.3 was reached. At OD600 0.3, 0.2% (w/v) l-arabinose was added to induce the expression of genes on the pDB1282 plasmid (except for BOLA3). At OD600 ∼ 0.6, 50 μM FeCl3 and 100 μM l-cysteine were added (except for BOLA3), and the culture was cooled in ice–water for 1 h with occasional shaking. Protein expression was induced by adding IPTG to a concentration of 0.2 mM, and incubation was continued at 18 °C with shaking at 180 rpm for an additional 12 h. The cells were harvested at 4 °C by centrifugation at 6000g for 15 min, flash-frozen in liquid nitrogen, and stored under liquid nitrogen until needed.

Protein Purification of BOLA3, ISCA1, ISCA2, ISCU, GLRX5, LIAS, and NFU1

The purification of each of the proteins was performed in an anaerobic chamber containing <1 ppm of O2 (Coy Laboratory products, Grass Lake, MI) by IMAC using the following general procedures with minor modifications for BOLA3. Cells were resuspended in 200 mL of lysis buffer (100 mM Tris–HCl, pH 8.0, 150 mM KCl, 10 mM imidazole, 10 mM BME, 10 mM MgCl2). To the resuspended cells, the following were added at their indicated final concentrations: 0.25 mM FeCl3, 1 mM l-cysteine, 1 mM PLP, one SIGMAFAST protease inhibitor tablet, 1 mM PMSF, 0.5 mg/mL lysozyme, and 0.01 mg/mL DNase (no FeCl3, cysteine, or PLP was added to BOLA3 samples). The cells were disrupted by sonication with an ultrasonic cell disruptor (Branson Sonifier II “model W-250”, Heinemann), and the lysates were clarified by centrifugation (4 °C, 45 000g, 1 h). The N-terminally His6-tagged SUMO fusion protein was then purified by Ni–NTA affinity chromatography. The Ni–NTA column was pre-equilibrated with 150 mL of lysis buffer. After loading the supernatant onto the Ni–NTA column, the resin was washed with 200 mL of wash buffer (50 mM HEPES, pH 7.5, 300 mM KCl, 30 mM imidazole, 10% glycerol (v/v), and 10 mM BME). Protein elution from the Ni–NTA resin was performed with 100 mL of elution buffer (50 mM HEPES, pH 7.5, 250 mM KCl, 300 mM imidazole, 10% glycerol, and 10 mM BME). The eluted protein was exchanged into cleavage buffer (50 mM HEPES, pH 7.5, 250 mM KCl, 5% glycerol, 40 mM imidazole, and 10 mM BME) using a PD-10 column. ULP1 protease (50 μg/mg of protein to be cleaved) was added to the fusion protein to excise the His6-SUMO tag from the protein of interest, and the reaction mixture was incubated on ice overnight. The following day, the protein sample was reloaded onto the Ni–NTA column pre-equilibrated in cleavage buffer, and the protein of interest was collected in the flow-through. The protein was concentrated to ∼2.5 mL and buffer-exchanged into storage buffer (50 mM HEPES, pH 7.5, 250 mM KCl, 30% glycerol, 2.5 mM TCEP, and 10 mM BME) using a PD-10 column (GE Healthcare). For proteins expressed from pET28a plasmid, their purification followed the same steps as described above, omitting the ULP1 cleavage step. When needed, the proteins were purified further by SEC on a HiPrep 16/60 Sephacryl HR S-200 column (Cytiva) equilibrated in gel filtration buffer (50 mM HEPES, pH 7.5, 250 mM KCl, 10% glycerol, 2.5 mM TCEP, and 10 mM BME) at a flow rate of 0.5 mL/min. (93) The S-200 column was connected to an AKTA protein liquid chromatography system (Cytiva) in an anaerobic chamber. Fractions containing the target protein were identified by UV–vis absorption at 280 nm and were combined, concentrated, and buffer-exchanged into storage buffer using a PD-10 column.
Amino acid analysis for LIAS and NFU1 was performed by the UC Davis Proteomics Core and revealed that the Bradford method overestimates the protein concentration of LIAS by a factor of 1.6 and that of NFU1 by a factor of 1.1. The concentration of the protein was determined by Bradford method using appropriate correction factors as necessary and using bovine serum albumin (fraction V) as the standard. (94) The purified protein sample was aliquoted, flash-frozen in liquid N2, and stored under liquid nitrogen until needed. Protein homogeneity was judged by 12% SDS–PAGE and was determined to be ≥95% pure. Colorimetric iron and sulfide analyses were conducted on the purified protein using the methods of Beinert. (95−97)

Overexpression and Purification of 57Fe-Labeled LIAS and NFU1 and Mössbauer Spectroscopy

To generate 57Fe-labeled proteins for analysis by Mössbauer spectroscopy, LIAS and NFUI proteins were overproduced as described above with the exception that they were supplemented with 50 μM 57FeCl3 instead of 50 μM FeCl3. The growth and purification procedures were essentially identical to those described above, with the exception that 57FeCl3 was also used for reconstitution during the lysis step. 57FeCl3 was prepared as previously described. (98)
For analysis by Mössbauer spectroscopy, 380 μM 57Fe-labeled LIAS or 860 μM NFU1 was loaded into Mössbauer cups and flash-frozen in liquid nitrogen. Mössbauer spectra were recorded on a spectrometer from SEECO (Edina, MN) equipped with a Janis SVT-400 variable-temperature cryostat. Isomer shifts are reported relative to the centroid of the spectrum of α-iron metal at room temperature. The external magnetic field was applied parallel to the direction of propagation of the γ radiation. The Mössbauer spectra were simulated using the WMOSS spectral analysis software from SEECO (www.wmoss.org, SEE Co., Edina, MN).

Overexpression and Purification of 34S-Labeled NFU1

The expression of apo-NFU1 was as reported for EcNfuA. (47) The protein was purified as described above with the exception that no FeCl3 or cysteine was added during the lysis step, as was done for the other proteins. The purified apo protein was chemically reconstituted with FeCl3 and Na234S in the same manner as was reported for EcNfuA. (47) The reconstituted NFU1 was centrifuged at 14 000g for 10 min to remove aggregates, concentrated to 2.5 mL, and buffer-exchanged into gel filtration buffer using a PD-10 column. The protein was then further purified on an S-200 SEC column as described above. The synthesis of Na234S followed the procedures previously reported. (47)

Electron Paramagnetic Resonance Spectroscopy Analysis of LIAS and NFUI

For analysis by EPR, 400 μM LIAS or NFU1 in storage buffer was prepared at 4 °C inside an anaerobic chamber. After a 15 min reduction with freshly prepared dithionite (4 mM final concentration), the samples were flash-frozen in cryogenic isopentane. Respective protein samples without dithionite were used as controls. Continuous-wave EPR spectra data were collected at 10 K with a microwave power of 10 mW and a modulation amplitude of 0.2 mT on a Magnettech 5000 X-band ESR spectrometer (Bruker) equipped with an ER 4102ST resonator. The temperature was controlled by an ER 4112-HV Oxford Instruments (Concord, MA) variable-temperature helium-151 flow cryostat.

Interaction between NFU1 and LIAS

Size-Exclusion Chromatography

The ability of NFU1 to interact with LIAS was investigated using size-exclusion gel filtration chromatography. To determine the association, 500 μL of each of the following protein samples was applied to a pre-equilibrated (gel filtration buffer) HiPrep 16/60 Sephacryl HR S-200 column (GE Healthcare) housed in a Coy anaerobic glovebox (<1 ppm of O2) and chromatographed using a flow rate of 0.5 mL/min. The elution volumes (Ve) of the following protein samples were determined: 100 μM LIAS alone, 200 μM NFU1 alone, and a 1:1 mixture of 200 μM LIAS plus 200 μM NFU1. For the molecular weight standard calibration curve, four individual injections were chromatographed for the standards as follows: a mixture of 250 μL of cytochrome c at 2 mg/mL (12.4 kDa) plus 250 μL of β-amylase at 4 mg/mL (200 kDa); a mixture of 250 μL of carbonic anhydrase at 3 mg/mL (29 kDa) plus 250 μL of alcohol dehydrogenase at 5 mg/mL (150 kDa); 500 μL of bovine serum albumin (66 kDa); 500 μL of blue dextran (2000 kDa). The elution volume of blue dextran was used for the void volume of the column (V0). The Ve of the standards was determined, and the calibration curve was plotted as the log of the molecular mass versus Ve/V0. The linear equation was then used to calculate the experimental molecular weight of each sample. The interaction was judged both by the calculated experimental size of each of the peaks as well as by a shift in the Ve. The presence of both LIAS and NFU1 was confirmed by SDS–PAGE.

Isothermal Titration Calorimetry

ITC was employed to confirm the interaction between LIAS and NFU1 observed using the gel filtration method above and to determine the binding dissociation constant (KD). The KD by ITC was determined by 2 μL injections of LIAS (150 μM in the syringe) into a solution of NFU1 (19 μM in the cell) at 27 °C using a MicroCal VP-ITC calorimeter (Malvern Pananalytical Ltd.) housed in an anaerobic chamber (<1 ppm of O2). Prior to the binding experiment, protein samples were thoroughly exchanged into ITC buffer (0.1 M HEPES pH 7.5, 0.5 M KCl, 5% glycerol, and 2 mM TCEP) by gel filtration chromatography using a PD-10 column. Binding analysis was accompanied by a control experiment in which LIAS ligand (150 μM) was titrated into the sample cell containing only the ITC buffer. Before the data were fit, the control raw data were subtracted from the corresponding raw titration data to account for the heat associated with ligand dilution. The corrected data were processed with the Origin 7 software package (Malvern Pananalytical Ltd.).

Liquid Chromatography–Mass Spectrometry Activity Assays

Activity measurements were conducted in a Coy anaerobic chamber. Each reaction mixture contained the following at their final concentrations (unless noted otherwise elsewhere): 50 mM HEPES, pH 7.5, 250 mM KCl, 10 μM LIAS, 10 μM SAH nucleosidase, 0.75 mM SAM, and 350 μM octanoyllysyl-containing peptide substrate [Glu-(N6-octanoyl)Lys-Ala-Tyr]. In reactions designed to assess the effect of BOLA3, ISCA1, ISCA2, ISCU, GLRX5, or NFUI, 200 μM (final concentration) of each protein was added individually or in combination as needed. For reactions in which GLRX5 was added, reduced glutathione was included to a final concentration of 1 mM. For reactions designed to test direct cluster transfer from NFU1 to LIAS during turnover, sodium citrate was included to final concentration of 5 mM. All reactions were initiated by the addition of 1 mM (final concentration) sodium dithionite, and 25 μL aliquots were removed at various times and added to an equal volume of quench solution that contained 300 mM H2SO4, 8 mM TCEP, and 10 μM AtsA peptide external standard. The samples were centrifuged at 14 000g for 30 min to remove any precipitated proteins. The time-dependent formation of 6-thiooctanoyl intermediate and lipoyl peptide products was determined by UPLC–MS/MS using multiple reaction monitoring (MRM) (Table S3). The quenched assay mixture was separated on an Agilent Technologies Zorbax Extend-C18 column Rapid Resolution HT (4.6 mm × 50 mm, 1.8 μm particle size) equilibrated in 98% solvent A (0.1% formic acid, pH 2.6) and 2% solvent B (100% acetonitrile). A solvent gradient of 2–65% B was applied from 0.5 to 2.5 min, the solvent composition was maintained at 65% B for 0.5 min before being returned to 2% B in 1 min, and then was retained at 2% B for an additional 1 min to re-equilibrate the column. A flow rate of 0.3 mL/min was maintained throughout the method (Table S4). The detection of the products was performed using electrospray ionization in positive mode (ESI+) with the following parameters: nitrogen gas temperature of 300 °C and flow rate of 5.0 L/min, nebulizer pressure of 15 psi, and capillary voltage of 4000 V. The products were quantified based on standard curves of product standards run under same conditions.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomedchemau.2c00020.

  • DNA and protein sequences, Mössbauer and EPR spectra, activity plots, and LC–MS conditions (PDF)

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Author Information

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  • Corresponding Authors
  • Authors
    • Douglas M. Warui - Department of Chemistry and and the , The Pennsylvania State University, University Park, Pennsylvania 16802, United States;  Orcidhttps://orcid.org/0000-0001-8950-9433
    • Debangsu Sil - Department of Chemistry and and the , The Pennsylvania State University, University Park, Pennsylvania 16802, United States;  Orcidhttps://orcid.org/0000-0002-0718-3925
    • Kyung-Hoon Lee - Department of Chemistry and and the , The Pennsylvania State University, University Park, Pennsylvania 16802, United States
    • Syam Sundar Neti - Department of Chemistry and and the , The Pennsylvania State University, University Park, Pennsylvania 16802, United States
    • Olga A. Esakova - Department of Chemistry and and the , The Pennsylvania State University, University Park, Pennsylvania 16802, United States;  Orcidhttps://orcid.org/0000-0001-8377-8368
    • Hayley L. Knox - Department of Chemistry and Biochemistry and Molecular Biology and the Howard Hughes Medical Institute, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by grants from the National Institutes of Health (awards GM-122595 to S.J.B. and GM-127079 to C.K.), the National Science Foundation (MCB-1716686 to S.J.B.), and the Eberly Family Distinguished Chair in Science (to S.J.B.). S.J.B. is an investigator of the Howard Hughes Medical Institute. This article is subject to HHMI’s Open Access to Publications policy. HHMI lab heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication.

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    Lipoic acid is a sulfur-contg. cofactor required for the function of several multienzyme complexes involved in the oxidative decarboxylation of α-keto acids and glycine. Mechanistic details of lipoic acid metab. are unclear in eukaryotes, despite two well defined pathways for synthesis and covalent attachment of lipoic acid in prokaryotes. Here, the involvement of four genes in the synthesis and attachment of lipoic acid in Saccharomyces cerevisiae is reported. LIP2 and LIP5 are required for lipoylation of all three mitochondrial target proteins: Lat1 and Kgd2, the resp. E2 subunits of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, and Gcv3, the H protein of the glycine cleavage enzyme. LIP3, which encodes a lipoate-protein ligase homolog, is necessary for lipoylation of Lat1 and Kgd2, and the enzymic activity of Lip3 is essential for this function. Finally, GCV3, encoding the H protein target of lipoylation, is itself absolutely required for lipoylation of Lat1 and Kgd2. It is shown that lipoylated Gcv3, and not glycine cleavage activity per se, is responsible for this function. Demonstration that a target of lipoylation is required for lipoylation is a novel result. Through anal. of the role of these genes in protein lipoylation, it is concluded that only one pathway for de novo synthesis and attachment of lipoic acid exists in yeast. A model for protein lipoylation is proposed in which Lip2, Lip3, Lip5, and Gcv3 function in a complex, which may be regulated by the availability of acetyl-CoA, and which in turn may regulate mitochondrial gene expression.
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  7. 7
    Spalding, M. D.; Prigge, S. T. Lipoic acid metabolism in microbial pathogens. Microbiology and molecular biology reviews: MMBR 2010, 74 (2), 200– 228,  DOI: 10.1128/MMBR.00008-10
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    Lipoic acid metabolism in microbial pathogens
    Spalding, Maroya D.; Prigge, Sean T.
    Microbiology and Molecular Biology Reviews (2010), 74 (2), 200-228CODEN: MMBRF7; ISSN:1092-2172. (American Society for Microbiology)
    A review. Lipoic acid [(R)-5-(1,2-dithiolan-3-yl)pentanoic acid] is an enzyme cofactor required for intermediate metab. in free-living cells. Lipoic acid was discovered nearly 60 years ago and was shown to be covalently attached to proteins in several multicomponent dehydrogenases. Cells can acquire lipoate (the deprotonated charge form of lipoic acid that dominates at physiol. pH) through either scavenging or de novo synthesis. Microbial pathogens implement these basic lipoylation strategies with a surprising variety of adaptations which can affect pathogenesis and virulence. Similarly, lipoylated proteins are responsible for effects beyond their classical roles in catalysis. These include roles in oxidative defense, bacterial sporulation, and gene expression. This review surveys the role of lipoate metab. in bacterial, fungal, and protozoan pathogens and how these organisms have employed this metab. to adapt to niche environments.
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  8. 8
    Cronan, J. E. Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation. EcoSal Plus 2014, 6 (1), 0001-2012,  DOI: 10.1128/ecosalplus.ESP-0001-2012
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  9. 9
    Ewald, R. Lipoate-Protein Ligase and Octanoyltransferase Are Essential for Protein Lipoylation in Mitochondria of Arabidopsis. Plant Physiology 2014, 165 (3), 978– 990,  DOI: 10.1104/pp.114.238311
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    9
    Lipoate-protein ligase and octanoyltransferase are essential for protein lipoylation in mitochondria of Arabidopsis
    Ewald, Ralph; Hoffmann, Christiane; Florian, Alexandra; Neuhaus, Ekkehard; Fernie, Alisdair R.; Bauwe, Hermann
    Plant Physiology (2014), 165 (3), 978-990CODEN: PLPHAY; ISSN:0032-0889. (American Society of Plant Biologists)
    Prosthetic lipoyl groups are required for the function of several essential multienzyme complexes, such as pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGDH), and the glycine cleavage system (glycine decarboxylase [GDC]). How these proteins are lipoylated has been extensively studied in prokaryotes and yeast (Saccharomyces cerevisiae), but little is known for plants. We earlier reported that mitochondrial fatty acid synthesis by ketoacyl-acyl carrier protein synthase is not vital for protein lipoylation in Arabidopsis (Arabidopsis thaliana) and does not play a significant role in roots. Here, we identify Arabidopsis lipoate-protein ligase (AtLPLA) as an essential mitochondrial enzyme that uses octanoyl-nucleoside monophosphate and possibly other donor substrates for the octanoylation of mitochondrial PDH-E2 and GDC H-protein; it shows no reactivity with bacterial and possibly plant KGDH-E2. The octanoate-activating enzyme is unknown, but we assume that it uses octanoyl moieties provided by mitochondrial β-oxidn. AtLPLA is essential for the octanoylation of PDH-E2, whereas GDC H-protein can optionally also be octanoylated by octanoyltransferase (LIP2) using octanoyl chains provided by mitochondrial ketoacyl-acyl carrier protein synthase to meet the high lipoate requirement of leaf mesophyll mitochondria. Similar to protein lipoylation in yeast, LIP2 likely also transfers octanoyl groups attached to the H-protein to KGDH-E2 but not to PDH-E2, which is exclusively octanoylated by LPLA. We suggest that LPLA and LIP2 together provide a basal protein lipoylation network to plants that is similar to that in other eukaryotes.
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  10. 10
    Cronan, J. E. Assembly of lipoic acid on its cognate enzymes: an extraordinary and essential biosynthetic pathway. Microbiol. Mol. Biol. Rev. 2016, 80, 429– 450,  DOI: 10.1128/MMBR.00073-15
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    10
    Assembly of lipoic acid on its cognate enzymes: an extraordinary and essential biosynthetic pathway
    Cronan, John E.
    Microbiology and Molecular Biology Reviews (2016), 80 (2), 429-450CODEN: MMBRF7; ISSN:1098-5557. (American Society for Microbiology)
    Although the structure of lipoic acid and its role in bacterial metab. were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become understood. Unlike most cofactors, lipoic acid must be covalently bound to its cognate enzyme proteins (the 2-oxoacid dehydrogenases and the glycine cleavage system) in order to function in central metab. Indeed, the cofactor is assembled on its cognate proteins rather than being assembled and subsequently attached as in the typical pathway, like that of biotin attachment. The first lipoate biosynthetic pathway detd. was that of Escherichia coli, which utilizes two enzymes to form the active lipoylated protein from a fatty acid biosynthetic intermediate. Recently, a more complex pathway requiring four proteins was discovered in Bacillus subtilis, which is probably an evolutionary relic. This pathway requires the H protein of the glycine cleavage system of single-carbon metab. to form active (lipoyl) 2-oxoacid dehydrogenases. The bacterial pathways inform the lipoate pathways of eukaryotic organisms. Plants use the E. coli pathway, whereas mammals and fungi probably use the B. subtilis pathway. The lipoate metab. enzymes (except those of sulfur insertion) are members of PFAM family PF03099 (the cofactor transferase family). Although these enyzmes share some sequence similarity, they catalyze three markedly distinct enzyme reactions, making the usual assignment of function based on alignments prone to frequent mistaken annotations. This state of affairs has possibly clouded the interpretation of one of the disorders of human lipoate metab.
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  11. 11
    Solmonson, A.; DeBerardinis, R. J. Lipoic acid metabolism and mitochondrial redox regulation. J. Biol. Chem. 2018, 293, 7522– 7530,  DOI: 10.1074/jbc.TM117.000259
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    11
    Lipoic acid metabolism and mitochondrial redox regulation
    Solmonson, Ashley; De Berardinis, Ralph J.
    Journal of Biological Chemistry (2018), 293 (20), 7522-7530CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    A review. Lipoic acid is an essential cofactor for mitochondrial metab. and is synthesized de novo using intermediates from mitochondrial fatty-acid synthesis type II, S-adenosylmethionine and iron-sulfur clusters. This cofactor is required for catalysis by multiple mitochondrial 2-ketoacid dehydrogenase complexes, including pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase. Lipoic acid also plays a crit. role in stabilizing and regulating these multienzyme complexes. Many of these dehydrogenases are regulated by reactive oxygen species, mediated through the disulfide bond of the prosthetic lipoyl moiety. Collectively, its functions explain why lipoic acid is required for cell growth, mitochondrial activity, and coordination of fuel metab.
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  12. 12
    Reed, L. J. A trail of research from lipoic acid to -keto acid dehydrogenase complexes. J. Biol. Chem. 2001, 276, 38329– 38336,  DOI: 10.1074/jbc.R100026200
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    12
    A trail of research from lipoic acid to α-keto acid dehydrogenase complexes
    Reed, Lester J.
    Journal of Biological Chemistry (2001), 276 (42), 38329-38336CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    A review, retracing the trail of research that began with the isolation and characterization of a microbial growth factor and led to elucidation of the structure, function, and regulation of α-keto acid dehydrogenase complexes.
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  13. 13
    Mayr, J. A.; Feichtinger, R. G.; Tort, F.; Ribes, A.; Sperl, W. Lipoic acid biosynthesis defects. J. Inherit. Metab. Dis. 2014, 37 (4), 553– 563,  DOI: 10.1007/s10545-014-9705-8
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    13
    Lipoic acid biosynthesis defects
    Mayr, Johannes A.; Feichtinger, Rene G.; Tort, Frederic; Ribes, Antonia; Sperl, Wolfgang
    Journal of Inherited Metabolic Disease (2014), 37 (4), 553-563CODEN: JIMDDP; ISSN:0141-8955. (Springer)
    A review. Lipoate is a covalently bound cofactor essential for five redox reactions in humans: in four 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). Two enzymes are from the energy metab., α-ketoglutarate dehydrogenase and pyruvate dehydrogenase; and three are from the amino acid metab., branched-chain ketoacid dehydrogenase, 2-oxoadipate dehydrogenase, and the GCS. All these enzymes consist of multiple subunits and share a similar architecture. Lipoate synthesis in mitochondria involves mitochondrial fatty acid synthesis up to octanoyl-acyl-carrier protein; and three lipoate-specific steps, including octanoic acid transfer to glycine cleavage H protein by lipoyl(octanoyl) transferase 2 (putative) (LIPT2), lipoate synthesis by lipoic acid synthetase (LIAS), and lipoate transfer by lipoyltransferase 1 (LIPT1), which is necessary to lipoylate the E2 subunits of the 2-oxoacid dehydrogenases. The reduced form dihydrolipoate is reactivated by dihydrolipoyl dehydrogenase (DLD). Mutations in LIAS have been identified that result in a variant form of nonketotic hyperglycinemia with early-onset convulsions combined with a defect in mitochondrial energy metab. with encephalopathy and cardiomyopathy. LIPT1 deficiency spares the GCS, and resulted in a combined 2-oxoacid dehydrogenase deficiency and early death in one patient and in a less severely affected individual with a Leigh-like phenotype. As LIAS is an iron-sulfur-cluster-dependent enzyme, a no. of recently identified defects in mitochondrial iron-sulfur cluster synthesis, including NFU1, BOLA3, IBA57, GLRX5 presented with deficiency of LIAS and a LIAS-like phenotype. As in DLD deficiency, a broader clin. spectrum can be anticipated for lipoate synthesis defects depending on which of the affected enzymes is most rate limiting.
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  14. 14
    Habarou, F.; Hamel, Y.; Haack, T. B.; Feichtinger, R. G.; Lebigot, E.; Marquardt Biallelic mutations in LIPT2 cause a mitochondrial lipoylation defect associated with severe neonatal encephalopathy. Am. J. Hum. Genet. 2017, 101, 283– 290,  DOI: 10.1016/j.ajhg.2017.07.001
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    14
    Biallelic Mutations in LIPT2 Cause a Mitochondrial Lipoylation Defect Associated with Severe Neonatal Encephalopathy
    Habarou, Florence; Hamel, Yamina; Haack, Tobias B.; Feichtinger, Rene G.; Lebigot, Elise; Marquardt, Iris; Busiah, Kanetee; Laroche, Cecile; Madrange, Marine; Grisel, Coraline; Pontoizeau, Clement; Eisermann, Monika; Boutron, Audrey; Chretien, Dominique; Chadefaux-Vekemans, Bernadette; Barouki, Robert; Bole-Feysot, Christine; Nitschke, Patrick; Goudin, Nicolas; Boddaert, Nathalie; Nemazanyy, Ivan; Delahodde, Agnes; Kolker, Stefan; Rodenburg, Richard J.; Korenke, G. Christoph; Meitinger, Thomas; Strom, Tim M.; Prokisch, Holger; Rotig, Agnes; Ottolenghi, Chris; Mayr, Johannes A.; de Lonlay, Pascale
    American Journal of Human Genetics (2017), 101 (2), 283-290CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
    Lipoate serves as a cofactor for the glycine cleavage system (GCS) and four 2-oxoacid dehydrogenases functioning in energy metab. (α-oxoglutarate dehydrogenase [α-KGDHc] and pyruvate dehydrogenase [PDHc]), or amino acid metab. (branched-chain oxoacid dehydrogenase, 2-oxoadipate dehydrogenase). Mitochondrial lipoate synthesis involves three enzymic steps catalyzed sequentially by lipoyl(octanoyl) transferase 2 (LIPT2), lipoic acid synthetase (LIAS), and lipoyltransferase 1 (LIPT1). Mutations in LIAS have been assocd. with nonketotic hyperglycinemia-like early-onset convulsions and encephalopathy combined with a defect in mitochondrial energy metab. LIPT1 deficiency spares GCS deficiency and has been assocd. with a biochem. signature of combined 2-oxoacid dehydrogenase deficiency leading to early death or Leigh-like encephalopathy. We report on the identification of biallelic LIPT2 mutations in three affected individuals from two families with severe neonatal encephalopathy. Brain MRI showed major cortical atrophy with white matter abnormalities and cysts. Plasma glycine was mildly increased. Affected individuals' fibroblasts showed reduced oxygen consumption rates, PDHc, α-KGDHc activities, leucine catabolic flux, and decreased protein lipoylation. A normalization of lipoylation was obsd. after expression of wild-type LIPT2, arguing for LIPT2 requirement in intramitochondrial lipoate synthesis. Lipoic acid supplementation did not improve clin. condition nor activities of PDHc, α-KGDHc, or leucine metab. in fibroblasts and was ineffective in yeast deleted for the orthologous LIP2.
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  15. 15
    Cao, X.; Zhu, L.; Song, X.; Hu, Z.; Cronan, J. E. Protein moonlighting elucidates the essential human pathway catalyzing lipoic acid assembly on its cognate enzymes. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (30), E7063– E7072,  DOI: 10.1073/pnas.1805862115
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    15
    Protein moonlighting elucidates the essential human pathway catalyzing lipoic acid assembly on its cognate enzymes
    Cao, Xinyun; Zhu, Lei; Song, Xuejiao; Hu, Zhe; Cronan, John E.
    Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (30), E7063-E7072CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The lack of attachment of lipoic acid to its cognate enzyme proteins results in devastating human metabolic disorders. These mitochondrial disorders are evident soon after birth and generally result in early death. The mutations causing specific defects in lipoyl assembly map in three genes, LIAS, LIPT1, and LIPT2. Although physiol. roles have been proposed for the encoded proteins, only the LIPT1 protein had been studied at the enzyme level. LIPT1 was reported to catalyze only the second partial reaction of the classical lipoate ligase mechanism. We report that the physiol. relevant LIPT1 enzyme activity is transfer of lipoyl moieties from the H protein of the glycine cleavage system to the E2 subunits of the 2-oxoacid dehydrogenases required for respiration (e.g., pyruvate dehydrogenase) and amino acid degrdn. We also report that LIPT2 encodes an octanoyl transferase that initiates lipoyl group assembly. The human pathway is now biochem. defined.
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  16. 16
    Parry, R. J. Biosynthesis of lipoic acid. 1. Incorporation of specifically tritiated octanoic acid into lipoic acid. J. Am. Chem. Soc. 1977, 99 (19), 6464– 6466,  DOI: 10.1021/ja00461a061
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    16
    Biosynthesis of lipoic acid. 1. Incorporation of specifically tritiated octanoic acid into lipoic acid
    Parry, Ronald J.
    Journal of the American Chemical Society (1977), 99 (19), 6464-6CODEN: JACSAT; ISSN:0002-7863.
    The mechanism of the conversion of octanoate into lipoate by Escherichia coli was examd. by administration of octanoic acid-carboxyl-14C to E. coli in conjunction with tritiated forms of octanoic acid labeled specifically at those C atoms which might be involved in the introduction of S. No 3H loss occurs from the methylene groups of octanoic acid adjacent to the sites of S introduction. Introduction of S at C-6 of octanoate proceeds with ∼50% 3H loss and introduction of S at C-8 of octanoate proceeds without significant loss of 3H.
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    White, R. H. Stable isotope studies on the biosynthesis of lipoic acid in Escherichia coli. Biochemistry 1980, 19 (1), 15– 19,  DOI: 10.1021/bi00542a003
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    17
    Stable isotope studies on the biosynthesis of lipoic acid in Escherichia coli
    White, Robert H.
    Biochemistry (1980), 19 (1), 15-19CODEN: BICHAW; ISSN:0006-2960.
    A method was developed for the gas chromatog.-mass spectrometric (GC-MS) identification of lipoic acid in tissue. The method consists of acid hydrolysis of the tissue to free the bound lipoic acid, methylene chloride extn. of the lipoic acid, and subsequent chem. derivatization of the lipoic acid as Me 6,8-bis(benzylthio)octanoate prior to GC-MS anal. By use of this method of anal., the incorporation of deuterium into lipoic acid by E. coli growing on acetate-methyl-2H3 was studied. The results showed that the lipoic acid was biosynthesized from octanoic acid with the loss of only 1 deuterium-contg. position at C-8. The deuterium incorporated at C-6 of octanoic acid from the labeled acetate was retained. Since this deuterium is incorporated in the L-configuration during fatty acid biosynthesis and is known to have the D-configuration in lipoic acid, it is concluded that an inversion of configuration occurs at C-6 during the sulfur insertion.
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    Jordan, S. W.; Cronan, J. E., Jr. A new metabolic link. J. Biol. Chem. 1997, 272, 17903– 17906,  DOI: 10.1074/jbc.272.29.17903
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    18
    A new metabolic link. The acyl carrier protein of lipid synthesis donates lipoic acid to the pyruvate dehydrogenase complex in Escherichia coli and mitochondria
    Jordan, Sean W.; Cronan, John E., Jr.
    Journal of Biological Chemistry (1997), 272 (29), 17903-17906CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Lipoic acid is an essential enzyme cofactor that requires covalent attachment to its cognate proteins to confer biol. activity. The major lipoylated proteins are highly conserved enzymes of central metab., the pyruvate and α-ketoglutarate dehydrogenase complexes. The classical lipoate ligase uses ATP to activate the lipoate carboxy group followed by attachment of the cofactor to a specific subunit of each dehydrogenase complex, and it was assumed that all lipoate attachment preceded by this mechanism. However, our previous work indicated in the absence of detectable ATP-dependent ligase activity raising the possibility of a class of enzyme that attaches lipoate to the dehydrogenase complexes by a different mechanism. We now report that E. coli and mitochondria contain lipoate transferases that use lipoyl-acyl carrier as the lipoate donor. This finding demonstrates a direct link between fatty acid synthesis and lipoate attachment an also provides the first direct demonstration of a role for the enigmatic acyl carrier proteins of mitochondria.
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    Jordan, S. W.; Cronan, J. E. [19] Biosynthesis of lipoic acid and posttranslational modification with lipoic acid in Escherichia coli. Methods Enzymol. 1997, 279, 176– 183,  DOI: 10.1016/S0076-6879(97)79021-9
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    19
    Biosynthesis of lipoic acid and posttranslational modification with lipoic acid in Escherichia coli
    Jordan, Sean W.; Cronan, John E., Jr.
    Methods in Enzymology (1997), 279 (Vitamins and Coenzymes, Part I), 176-183CODEN: MENZAU; ISSN:0076-6879. (Academic)
    A report is given on the biosynthetic pathway of lipoic acid and the posttranslational modification of proteins by lipoate ligases in E. coli.
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  20. 20
    Jordan, S. W.; Cronan, J. E. The Escherichia coli lipB Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase. J. Bacteriol. 2003, 185 (5), 1582– 1589,  DOI: 10.1128/JB.185.5.1582-1589.2003
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    20
    The Escherichia coli lipB gene encodes lipoyl (octanoyl)-acyl carrier protein:protein transferase
    Jordan, Sean W.; Cronan, John E., Jr.
    Journal of Bacteriology (2003), 185 (5), 1582-1589CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    In an earlier study we reported a new enzyme, lipoyl-[acyl carrier protein]-protein N-lipoyltransferase, in Escherichia coli and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase. It was also shown that E. coli lipB mutants lack this enzyme activity, a finding consistent with lipB being the gene that encoded the lipoyltransferase. However, it remained possible that lipB encoded a pos. regulator required for lipoyltransferase expression or action. We now report genetic and biochem. evidence demonstrating that lipB encodes the lipoyltransferase. A lipB temp.-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the lipB-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. In the course of these expts. the ATG initiation codon commonly assigned to lipB genes in genomic databases was shown to produce a nonfunctional E. coli LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymically active protein. Prior genetic results suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addn. to its normal substrates lipoic acid plus ATP. We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. A nonhydrolyzable lipoyl-AMP analog was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chem. diverse reactions.
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    Nesbitt, N. M. Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase. Protein Expr Purif 2005, 39 (2), 269– 82,  DOI: 10.1016/j.pep.2004.10.021
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    Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase
    Nesbitt, Natasha M.; Baleanu-Gogonea, Camelia; Cicchillo, Robert M.; Goodson, Kathy; Iwig, David F.; Broadwater, John A.; Haas, Jeffrey A.; Fox, Brian G.; Booker, Squire J.
    Protein Expression and Purification (2005), 39 (2), 269-282CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier)
    Lipoic acid is a sulfur-contg. 8-carbon fatty acid that functions as a central cofactor in multienzyme complexes that are involved in the oxidative decarboxylation of glycine and several α-keto acids. In its functional form, it is bound covalently in an amide linkage to the ε-amino group of a conserved lysine residue of the "lipoyl bearing subunit," resulting in a long "swinging arm" that shuttles intermediates among the requisite active sites. In Escherichia coli and many other organisms, the lipoyl cofactor can be synthesized endogenously. The 8-carbon fatty-acyl chain is constructed via the type II fatty acid biosynthetic pathway as an appendage to the acyl carrier protein (ACP). Lipoyl(octanoyl)transferase (LipB) transfers the octanoyl chain from ACP to the target lysine acceptor, generating the substrate for lipoyl synthase (LS), which subsequently catalyzes insertion of both sulfur atoms into the C-6 and C-8 positions of the octanoyl chain. In this study, we present a three-step isolation procedure that results in a 14-fold purifn. of LipB to >95% homogeneity in an overall yield of 25%. We also show that the protein catalyzes the transfer of the octanoyl group from octanoyl-ACP to apo-H protein, which is the lipoyl bearing subunit of the glycine cleavage system. The specific activity of the purified protein is 0.541 U mg-1, indicating a turnover no. of ∼0.2 s-1, and the apparent Km values for octanoyl-ACP and apo-H protein are 10.2±4.4 and 13.2±2.9 μM, resp.
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    Parry, R. J.; Trainor, D. A. Biosynthesis of lipoic acid. 2. Stereochemistry of sulfur introduction at C-6 of octanoic acid. J. Am. Chem. Soc. 1978, 100 (16), 5243– 5244,  DOI: 10.1021/ja00484a073
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    22
    Biosynthesis of lipoic acid. 2. Stereochemistry of sulfur introduction at C-6 of octanoic acid
    Parry, Ronald J.; Trainor, Diane A.
    Journal of the American Chemical Society (1978), 100 (16), 5243-4CODEN: JACSAT; ISSN:0002-7863.
    (6R)- and (6S)-Octanoic acid-3H were synthesized and their incorporation into lipoic acid by Escherichia coli was investigated. The precursor incorporation expts. demonstrated that the 6-pro-R H atom of octanoic acid is lost as the result of S atom introduction and that the S atom introduction therefore proceeds with inversion of configuration.
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    Reed, K. E.; Cronan, J. E. Lipoic acid metabolism in Escherichia coli: sequencing and functional characterization of the lipA and lipB genes. J. Bacteriol. 1993, 175 (5), 1325– 1336,  DOI: 10.1128/jb.175.5.1325-1336.1993
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    23
    Lipoic acid metabolism in Escherichia coli: Sequencing and functional characterization of the lipA and lipB genes
    Reed, Kelynne E.; Cronan, John E., Jr.
    Journal of Bacteriology (1993), 175 (5), 1325-36CODEN: JOBAAY; ISSN:0021-9193.
    Two genes, lipA and lipB, involved in lipoic acid biosynthesis or metab. were characterized by DNA sequence anal. The translational initiation site of the lipA gene was established, and the lipB gene product was identified as a 15-kDa protein. Overprodn. of LipA resulted in the formation of inclusion bodies, from which the protein was readily purified. Cells grown under strictly anaerobic conditions required the lipA and lipB gene products for the synthesis of a functional glycine cleavage system. Mutants carrying a null mutation in the lipB gene retained a partial ability to synthesize lipoic acid and produced low levels of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase activities. The lipA gene product failed to convert protein-bound octanoic acid moieties to lipoic acid moieties in vivo; however, the growth of both lipA and lipB mutants was supported by either 6-thiooctanoic acid or 8-thiooctanoic acid in place of lipoic acid. These data suggest that LipA is required from the insertion of the first sulfur into the octanoic acid backbone. LipB functions downstream of LipA, but its role in lipoic acid metab. remains unclear.
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    Miller, J. R. Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein. Biochemistry 2000, 39, 15166– 15178,  DOI: 10.1021/bi002060n
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    Escherichia coli LipA Is a Lipoyl Synthase: In Vitro Biosynthesis of Lipoylated Pyruvate Dehydrogenase Complex from Octanoyl-Acyl Carrier Protein
    Miller, J. Richard; Busby, Robert W.; Jordan, Sean W.; Cheek, Jennifer; Henshaw, Timothy F.; Ashley, Gary W.; Broderick, Joan B.; Cronan, John E., Jr.; Marletta, Michael A.
    Biochemistry (2000), 39 (49), 15166-15178CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been obsd. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as sol. proteins. The proteins were purified as a mixt. of monomeric and dimeric species that contain approx. four iron atoms per LipA polypeptide and a similar amt. of acid-labile sulfide. ESR and electronic absorbance spectroscopy indicate that the proteins contain a mixt. of [3Fe-4S] and [4Fe-4S] cluster states. Redn. with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S]1+ cluster with the majority of the protein contg. a species consistent with an S = 0 [4Fe-4S]2+ cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), resp., to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chem.
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    Cicchillo, R. M. Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid. Biochemistry 2004, 43 (21), 6378– 86,  DOI: 10.1021/bi049528x
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    Lipoyl Synthase Requires Two Equivalents of S-Adenosyl-L-methionine To Synthesize One Equivalent of Lipoic Acid
    Cicchillo, Robert M.; Iwig, David F.; Jones, A. Daniel; Nesbitt, Natasha M.; Baleanu-Gogonea, Camelia; Souder, Matthew G.; Tu, Loretta; Booker, Squire J.
    Biochemistry (2004), 43 (21), 6378-6386CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various α-keto acids, as well as the cleavage of glycine into CO2 and NH3, with concomitant transfer of its α-carbon to tetrahydrofolate, generating N5,N10-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochem. studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-L-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA•), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming L-methionine in addn. to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated deriv. of the lipoyl-bearing subunit (E2) of the pyruvate dehydrogenase complex. Herein, the authors show that the octanoylated deriv. of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, the authors show that the 5'-dA• acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d15]H-protein to 5'-deoxyadenosine. Last, the authors' data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.
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    Oberg, N. RadicalSAM.org: A Resource to Interpret Sequence-Function Space and Discover New Radical SAM Enzyme Chemistry. ACS Bio & Med. Chem. Au 2022, 2 (1), 22– 35,  DOI: 10.1021/acsbiomedchemau.1c00048
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    RadicalSAM.org: A Resource to Interpret Sequence-Function Space and Discover New Radical SAM Enzyme Chemistry
    Oberg, Nils; Precord, Timothy W.; Mitchell, Douglas A.; Gerlt, John A.
    ACS Bio & Med Chem Au (2022), 2 (1), 22-35CODEN: ABMCB8; ISSN:2694-2437. (American Chemical Society)
    The radical SAM superfamily (RSS), arguably the most functionally diverse enzyme superfamily, is also one of the largest with ~ 700 K members currently in the UniProt database. The vast majority of the members have uncharacterized enzymic activities and metabolic functions. In this Perspective, we describe RadicalSAM.org, a new web-based resource that enables a user-friendly genomic enzymol. strategy to explore sequence-function space in the RSS. The resource attempts to enable identification of isofunctional groups of radical SAM enzymes using sequence similarity networks (SSNs) and the genome context of the bacterial, archaeal, and fungal members provided by genome neighborhood diagrams (GNDs). Enzymic activities and in vivo functions frequently can be inferred from genome context given the tendency for genes of related function to be clustered. We invite the scientific community to use RadicalSAM.org to (i) guide their exptl. studies to discover new enzymic activities and metabolic functions, (ii) contribute exptl. verified annotations to RadicalSAM.org to enhance the ability to predict novel activities and functions, and (iii) provide suggestions for improving this resource.
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    Broderick, J. B. Radical S-Adenosylmethionine Enzymes. Chem. Rev. 2014, 114 (8), 4229– 4317,  DOI: 10.1021/cr4004709
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    Radical S-adenosylmethionine enzymes
    Broderick, Joan B.; Duffus, Benjamin R.; Duschene, Kaitlin S.; Shepard, Eric M.
    Chemical Reviews (Washington, DC, United States) (2014), 114 (8), 4229-4317CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. The review begins by summarizing unifying features of radical S-adenosylmethionine (SAM) enzymes, and subsequent sections delve into the biochem., spectroscopic, structural, and mechanistic details for those enzymes that have been characterized.
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    Landgraf, B. J.; McCarthy, E. L.; Booker, S. J. Radical S-Adenosylmethionine Enzymes in Human Health and Disease. Annu. Rev. Biochem. 2016, 85 (1), 485– 514,  DOI: 10.1146/annurev-biochem-060713-035504
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    Radical S-Adenosylmethionine Enzymes in Human Health and Disease
    Landgraf, Bradley J.; McCarthy, Erin L.; Booker, Squire J.
    Annual Review of Biochemistry (2016), 85 (), 485-514CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
    A review. Radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chem. challenging reactions across all domains of life. Of approx. 114,000 of these enzymes, 8 are known to be present in humans: MOCS1, molybdenum cofactor biosynthesis; LIAS, lipoic acid biosynthesis; CDK5RAP1, 2-methylthio-N6-isopentenyladenosine biosynthesis; CDKAL1, methylthio-N6-threonylcarbamoyladenosine biosynthesis; TYW1, wybutosine biosynthesis; ELP3, 5-methoxycarbonylmethyl uridine; and RSAD1 and viperin, both of unknown function. Aberrations in the genes encoding these proteins result in a variety of diseases. In this review, we summarize the biochem. characterization of these 8 radical S-adenosylmethionine enzymes and, in the context of human health, describe the deleterious effects that result from such genetic mutations.
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    Holliday, G. L. Atlas of the Radical SAM Superfamily: Divergent Evolution of Function Using a ″Plug and Play″ Domain. Methods Enzymol 2018, 606, 1– 71,  DOI: 10.1016/bs.mie.2018.06.004
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    Atlas of the radical SAM superfamily: divergent evolution of function using a "plug and play" domain
    Holliday, Gemma L.; Akiva, Eyal; Meng, Elaine C.; Brown, Shoshana D.; Calhoun, Sara; Pieper, Ursula; Sali, Andrej; Booker, Squire J.; Babbitt, Patricia C.
    Methods in Enzymology (2018), 606 (Radical SAM Enzymes), 1-71CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)
    The radical SAM superfamily contains over 100,000 homologous enzymes that catalyze a remarkably broad range of reactions required for life, including metab., nucleic acid modification, and biogenesis of cofactors. While the highly conserved SAM-binding motif responsible for formation of the key 5'-deoxyadenosyl radical intermediate is a key structural feature that simplifies identification of superfamily members, our understanding of their structure-function relationships is complicated by the modular nature of their structures, which exhibit varied and complex domain architectures. To gain new insight about these relationships, we classified the entire set of sequences into similarity-based subgroups that could be visualized using sequence similarity networks. This superfamily-wide anal. reveals important features that had not previously been appreciated from studies focused on one or a few members. Functional information mapped to the networks indicates which members have been exptl. or structurally characterized, their known reaction types, and their phylogenetic distribution. Despite the biol. importance of radical SAM chem., the vast majority of superfamily members have never been exptl. characterized in any way, suggesting that many new reactions remain to be discovered. In addn. to 20 subgroups with at least one known function, we identified addnl. subgroups made up entirely of sequences of unknown function. Importantly, our results indicate that even general reaction types fail to track well with our sequence similarity-based subgroupings, raising major challenges for function prediction for currently identified and new members that continue to be discovered. Interactive similarity networks and other data from this anal. are available from the Structure-Function Linkage Database.
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    Booker, S. J.; Grove, T. L. Mechanistic and functional versatility of radical SAM enzymes. F1000 Biol. Rep. 2010, 2, 52,  DOI: 10.3410/B2-52
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    Mechanistic and functional versatility of radical SAM enzymes
    Booker Squire J; Grove Tyler L
    F1000 biology reports (2010), 2 (), 52 ISSN:.
    Enzymes of the radical SAM (RS) superfamily catalyze a diverse assortment of reactions that proceed via intermediates containing unpaired electrons. The radical initiator is the common metabolite S-adenosyl-l-methionine (SAM), which is reductively cleaved to generate a 5'-deoxyadenosyl 5'-radical, a universal and obligate intermediate among enzymes within this class. A bioinformatics study that appeared in 2001 indicated that this superfamily contained over 600 members, many catalyzing reactions that were rich in novel chemical transformations. Since that seminal study, the RS superfamily has grown immensely, and new details about the scope of reactions and biochemical pathways in which its members participate have emerged. This review will highlight only a few of the most significant findings from the past 2-3 years, focusing primarily on: RS enzymes involved in complex metallocofactor maturation; characterized RS enzymes that lack the canonical CxxxCxxC motif; RS enzymes containing multiple iron-sulfur clusters; RS enzymes catalyzing reactions with compelling medical implications; and the energetics and mechanism of generating the 5'-deoxyadenosyl radical. A number of significant studies of RS enzymes will unfortunately be omitted, and it is hoped that the reader will access the relevant literature - particularly a number of superb review articles recently written on the subject - to acquire a deeper appreciation of this class of enzymes.
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    Bandarian, V. Journey on the Radical SAM Road as an Accidental Pilgrim. ACS Bio Med Chem Au 2022,  DOI: 10.1021/acsbiomedchemau.1c00059
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    Frey, P. A.; Booker, S. J. Radical mechanisms of S-adenosylmethionine-dependent enzymes. Adv. Protein Chem. 2001, 58, 1– 45,  DOI: 10.1016/S0065-3233(01)58001-8
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    Radical mechanisms of S-adenosylmethionine-dependent enzymes
    Frey, Perry A.; Booker, Squire J.
    Advances in Protein Chemistry (2001), 58 (Novel Cofactors), 1-45CODEN: APCHA2; ISSN:0065-3233. (Academic Press)
    A review. The authors describe the family of S-adenosylmethionine (SAM)-dependent enzymes that make use of the 5'-deoxyadenosyl radical, with special ref. to the mechanism by which SAM is cleaved reversibly at the active site. They also consider the functions of the 5'-deoxyadenosyl radical and the mechanisms of these diverse reactions. Special consideration is given to lysine 2,3-aminomutase, pyruvate formate lyase, anaerobic ribonucleotide reductase and biotin synthase. (c) 2001 Academic Press.
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    Frey, P. A.; Hegeman, A. D.; Ruzicka, F. J. The Radical SAM Superfamily. Crit Rev. Biochem Mol. Biol. 2008, 43 (1), 63– 88,  DOI: 10.1080/10409230701829169
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    The Radical SAM Superfamily
    Frey, Perry A.; Hegeman, Adrian D.; Ruzicka, Frank J.
    Critical Reviews in Biochemistry and Molecular Biology (2008), 43 (1), 63-88CODEN: CRBBEJ; ISSN:1040-9238. (Informa Healthcare)
    A review. The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The characteristic three-cysteine motif nucleates a [4Fe-4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochem. transformations, and most members have not been biochem. characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochem. information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochem. characterized to date have in common the cleavage of the [4Fe-4S]1 + -SAM complex to [4Fe-4S]2 +-Met and the 5' -deoxyadenosyl radical, which abstrs. a hydrogen atom from the substrate to initiate a radical mechanism.
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    Cicchillo, R. M. Escherichia coli lipoyl synthase binds two distinct [4Fe–4S] clusters per polypeptide. Biochemistry 2004, 43, 11770– 11781,  DOI: 10.1021/bi0488505
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    Escherichia coli Lipoyl Synthase Binds Two Distinct [4Fe-4S] Clusters per Polypeptide
    Cicchillo, Robert M.; Lee, Kyung-Hoon; Baleanu-Gogonea, Camelia; Nesbitt, Natasha M.; Krebs, Carsten; Booker, Squire J.
    Biochemistry (2004), 43 (37), 11770-11781CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-L-methionine (SAM) as the precursor to a high-energy 5'-deoxyadenosyl 5'-radical (5'-dA•). In the LS reaction, the 5'-dA• is hypothesized to abstr. hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM are required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d15 H-protein of the glycine cleavage system-one of the substrates for LS-has been reported. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X3-C-X2-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X4-C-X5-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 ± 0.5 irons and 6.4 ± 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5'-deoxyadenosine (5'-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 ± 0.1 irons and 3.6 ± 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 ± 0.1 irons and 4.7 ± 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5'-dA or the lipoyl group. Moessbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all assocd. iron is in the configuration [4Fe-4S]2+. When wild-type LS was reconstituted with 57Fe and sodium sulfide, it harbored considerably more iron (13.8 ± 0.6) and sulfide (13.1 ± 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5'-dA and 0.36 equiv of the lipoyl group. Moessbauer spectroscopy of this protein revealed that only ∼67% ± 6% of the iron is in the form of [4Fe-4S]2+ clusters, amounting to 9.2 ± 0.4 irons and 8.8 ± 0.1 sulfides or 2 [4Fe-4S]2+ clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the Moessbauer parameters of the clusters assocd. with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concn. and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-contg. motifs.
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    Douglas, P. Lipoyl synthase inserts sulfur atoms into an octanoyl substrate in a stepwise manner. Angew. Chem., Int. Ed. Engl. 2006, 45 (31), 5197– 9,  DOI: 10.1002/anie.200601910
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    Lipoyl synthase inserts sulfur atoms into an octanoyl substrate in a stepwise manner
    Douglas Paul; Kriek Marco; Bryant Penny; Roach Peter L
    Angewandte Chemie (International ed. in English) (2006), 45 (31), 5197-9 ISSN:1433-7851.
    There is no expanded citation for this reference.
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    Lanz, N. D. Evidence for a catalytically and kinetically competent enzyme-substrate cross-linked intermediate in catalysis by lipoyl synthase. Biochemistry 2014, 53, 4557– 4572,  DOI: 10.1021/bi500432r
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    Evidence for a Catalytically and Kinetically Competent Enzyme-Substrate Cross-Linked Intermediate in Catalysis by Lipoyl Synthase
    Lanz, Nicholas D.; Pandelia, Maria-Eirini; Kakar, Elizabeth S.; Lee, Kyung-Hoon; Krebs, Carsten; Booker, Squire J.
    Biochemistry (2014), 53 (28), 4557-4572CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Lipoyl synthase (LS) catalyzes the final step in lipoyl cofactor biosynthesis: the insertion of two sulfur atoms at C6 and C8 of an (N6-octanoyl)-lysyl residue on a lipoyl carrier protein (LCP). LS is a member of the radical SAM superfamily, enzymes that use a [4Fe-4S] cluster to effect the reductive cleavage of S-adenosyl-L-methionine (SAM) to L-methionine and a 5'-deoxyadenosyl 5'-radical (5'-dA•). In the LS reaction, two equiv. of 5'-dA• are generated sequentially to abstr. hydrogen atoms from C6 and C8 of the appended octanoyl group, initiating sulfur insertion at these positions. The second [4Fe-4S] cluster on LS, termed the auxiliary cluster, is proposed to be the source of the inserted sulfur atoms. Herein, we provide evidence for the formation of a covalent cross-link between LS and an LCP or synthetic peptide substrate in reactions in which insertion of the second sulfur atom is slowed significantly by deuterium substitution at C8 or by inclusion of limiting concns. of SAM. The observation that the proteins elute simultaneously by anion-exchange chromatog. but are sepd. by aerobic SDS-PAGE is consistent with their linkage through the auxiliary cluster that is sacrificed during turnover. Generation of the cross-linked species with a small, unlabeled (N6-octanoyl)-lysyl-contg. peptide substrate allowed demonstration of both its chem. and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction. Mossbauer spectroscopy of the cross-linked intermediate reveals that one of the [4Fe-4S] clusters, presumably the auxiliary cluster, is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to those of reduced [3Fe-4S]0 clusters.
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    Lanz, N. D. Characterization of a radical intermediate in lipoyl cofactor biosynthesis. J. Am. Chem. Soc. 2015, 137, 13216– 13219,  DOI: 10.1021/jacs.5b04387
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    Characterization of a radical intermediate in lipoyl cofactor biosynthesis
    Lanz, Nicholas D.; Rectenwald, Justin M.; Wang, Bo; Kakar, Elizabeth S.; Laremore, Tatiana N.; Booker, Squire J.; Silakov, Alexey
    Journal of the American Chemical Society (2015), 137 (41), 13216-13219CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Lipoyl synthase (LipA) catalyzes the final step in the biosynthesis of lipoyl cofactor, the insertion of 2 sulfur atoms at C6 and C8 of an n-octanoyl chain. LipA is a member of the radical S-adenosylmethionine (SAM) superfamily of enzymes and uses 2 [4Fe-4S] clusters to catalyze its transformation. One cluster binds in contact with SAM and donates the requisite electron for the reductive cleavage of SAM to generate 2 5'-deoxyadenosyl 5'-radicals, which abstr. H atoms from C6 and C8 of the substrate. By contrast, the 2nd, auxiliary [4Fe-4S] cluster, has been hypothesized to serve as the sulfur donor in the reaction. Such a sacrificial role for an Fe-S cluster during catalysis has not been universally accepted. The use of a conjugated 2,4-hexadienoyl-contg. substrate analog has allowed the substrate radical to be trapped and characterized by continuous-wave and pulsed ESR methods. Here, the authors report the observation of a 57Fe hyperfine coupling interaction with the paramagnetic signal, which indicates that the Fe-S cluster of LipA and its substrate are within bonding distance.
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    Lanz, N. D.; Booker, S. J. Auxiliary iron-sulfur cofactors in radical SAM enzymes. Biochim. Biophys. Acta 2015, 1853 (6), 1316– 34,  DOI: 10.1016/j.bbamcr.2015.01.002
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    38
    Auxiliary iron-sulfur cofactors in radical SAM enzymes
    Lanz, Nicholas D.; Booker, Squire J.
    Biochimica et Biophysica Acta, Molecular Cell Research (2015), 1853 (6), 1316-1334CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
    A review. A vast no. of enzymes are now known to belong to a superfamily known as radical SAM, which all contain a [4Fe-4S] cluster ligated by three cysteine residues. The remaining, unligated, iron ion of the cluster binds in contact with the α-amino and α-carboxylate groups of S-adenosyl-L-methionine (SAM). This binding mode facilitates inner-sphere electron transfer from the reduced form of the cluster into the sulfur atom of SAM, resulting in a reductive cleavage of SAM to methionine and a 5'-deoxyadenosyl radical. The 5'-deoxyadenosyl radical then abstrs. a target substrate hydrogen atom, initiating a wide variety of radical-based transformations. A subset of radical SAM enzymes contains one or more addnl. iron-sulfur clusters that are required for the reactions they catalyze. However, outside of a subset of sulfur insertion reactions, very little is known about the roles of these addnl. clusters. This review will highlight the most recent advances in the identification and characterization of radical SAM enzymes that harbor auxiliary iron-sulfur clusters. This article is part of a Special Issue entitled: Fe/S proteins: Anal., structure, function, biogenesis and diseases.
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  39. 39
    Lanz, N. D.; Booker, S. J. The role of iron-sulfur clusters in the biosynthesis of the lipoyl cofactor. In Iron-Sulfur Clusters in Chemistry and Biology; Rouault, T. A., Ed.; Walter de Gruyter GMbH: Berlin, Germany, 2014.
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    Lanz, N. D.; Booker, S. J. Identification and function of auxiliary iron-sulfur clusters in radical SAM enzymes. Biochim. Biophys. Acta 2012, 1824 (11), 1196– 212,  DOI: 10.1016/j.bbapap.2012.07.009
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    40
    Identification and function of auxiliary iron-sulfur clusters in radical SAM enzymes
    Lanz, Nicholas D.; Booker, Squire J.
    Biochimica et Biophysica Acta, Proteins and Proteomics (2012), 1824 (11), 1196-1212CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)
    A review. Radical SAM (RS) enzymes use a 5'-deoxyadenosyl 5'-radical generated from a reductive cleavage of S-adenosyl-L-methionine to catalyze over 40 distinct reaction types. A distinguishing feature of these enzymes is a [4Fe-4S] cluster to which each of three iron ions is ligated by three cysteinyl residues most often located in a Cx3Cx2C motif. The α-amino and α-carboxylate groups of SAM anchor the mol. to the remaining iron ion, which presumably facilitates its reductive cleavage. A subset of RS enzymes contains addnl. iron-sulfur clusters, - which we term auxiliary clusters - most of which have unidentified functions. Enzymes in this subset are involved in cofactor biosynthesis and maturation, post-transcriptional and post-translational modification, enzyme activation, and antibiotic biosynthesis. The addnl. clusters in these enzymes have been proposed to function in sulfur donation, electron transfer, and substrate anchoring. This review will highlight evidence supporting the presence of multiple iron-sulfur clusters in these enzymes as well as their predicted roles in catalysis.
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  41. 41
    Walsby, C. J. An anchoring role for FeS clusters: chelation of the amino acid moiety of S-adenosylmethionine to the unique iron site of the [4Fe–4S] cluster of pyruvate formate–lyase activating enzyme. J. Am. Chem. Soc. 2002, 124, 11270– 11271,  DOI: 10.1021/ja027078v
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    41
    An Anchoring Role for FeS Clusters: Chelation of the Amino Acid Moiety of S-Adenosylmethionine to the Unique Iron Site of the [4Fe-4S] Cluster of Pyruvate Formate-Lyase Activating Enzyme
    Walsby, Charles J.; Ortillo, Danilo; Broderick, William E.; Broderick, Joan B.; Hoffman, Brian M.
    Journal of the American Chemical Society (2002), 124 (38), 11270-11271CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Pyruvate formate-lyase activating enzyme (PFL-AE) generates the catalytically essential glycyl radical on pyruvate formate-lyase via the interaction of the catalytically active (4Fe-4S)+ cluster with S-adenosylmethionine (AdoMet). Like other members of the Fe-S/AdoMet family of enzymes, PFL-AE is thought to function via generation of an AdoMet-derived 5'-deoxyadenosyl radical intermediate; however, the mechanistic steps by which this radical is generated remain to be elucidated. While all of the members of the Fe-S/AdoMet family of enzymes appear to have a unique iron site in the (4Fe-4S) cluster, based on the presence of a conserved three-cysteine cluster binding motif, the role of this unique site has been elusive. Here we utilize 35-GHz pulsed electron nuclear double resonance (ENDOR) studies of the (4Fe-4S)+ cluster of PFL-AE in complex with isotopically labeled AdoMet [denoted (1+/AdoMet)] to show that the unique iron serves to anchor the AdoMet for catalysis. AdoMet labeled with 17O at the carboxylate shows a coupling of A = 12.2 MHz, consistent with direct coordination of the carboxylate to the unique iron of the cluster. This is supported by 13C-ENDOR with the carboxylato carbon labeled with 13C, which shows a hyperfine coupling of 0.71 MHz. AdoMet enriched with 15N at the amino position gives rise to a spectrum with A(15N) = 5.8 MHz, consistent with direct coordination of the amino group to a unique iron of the cluster. Together, the results demonstrate that the unique iron of the (4Fe-4S) cluster anchors AdoMet by forming a classical N/O chelate with the amino and carboxylato groups of the methionine fragment.
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    Walsby, C. J. Electron-nuclear double resonance spectroscopic evidence that S-adenosylmethionine binds in contact with the catalytically active [4Fe-4S]+ cluster of pyruvate formate-lyase activating enzyme. J. Am. Chem. Soc. 2002, 124, 3143– 3151,  DOI: 10.1021/ja012034s
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    42
    Electron-Nuclear Double Resonance Spectroscopic Evidence That S-Adenosylmethionine Binds in Contact with the Catalytically Active [4Fe-4S]+ Cluster of Pyruvate Formate-Lyase Activating Enzyme
    Walsby, Charles J.; Hong, Wei; Broderick, William E.; Cheek, Jennifer; Ortillo, Danilo; Broderick, Joan B.; Hoffman, Brian M.
    Journal of the American Chemical Society (2002), 124 (12), 3143-3151CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Pyruvate formate-lyase activating enzyme (PFL-AE) is a representative member of an emerging family of enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (AdoMet) to initiate radical catalysis. Although these enzymes have diverse functions, evidence is emerging that they operate by a common mechanism in which a [4Fe-4S]+ interacts with AdoMet to generate a 5'-deoxyadenosyl radical intermediate. To date, however, it has been unclear whether the iron-sulfur cluster is a simple electron-transfer center or whether it participates directly in the radical generation chem. Here we utilize ESR (EPR) and pulsed 35 GHz electron-nuclear double resonance (ENDOR) spectroscopy to address this question. EPR spectroscopy reveals a dramatic effect of AdoMet on the EPR spectrum of the [4Fe-4S]+ of PFL-AE, changing it from rhombic (g = 2.02, 1.94, 1.88) to nearly axial (g = 2.01, 1.88, 1.87). 2H and 13C ENDOR spectroscopy was performed on [4Fe-4S]+-PFL-AE (S = 1/2) in the presence of AdoMet labeled at the Me position with either 2H or 13C (denoted [1+/AdoMet]). The observation of a substantial 2H coupling of ∼1 MHz (∼6-7 MHz for 1H), as well as hyperfine-split signals from the 13C, manifestly require that AdoMet lie close to the cluster. 2H and 13C ENDOR data were also obtained for the interaction of AdoMet with the diamagnetic [4Fe-4S]2+ state of PFL-AE, which is visualized through cryoredn. of the frozen [4Fe-4S]2+/AdoMet complex to form the reduced state (denoted [2+/AdoMet]red) trapped in the structure of the oxidized state. 2H and 13C ENDOR spectra for [2+/AdoMet]red are essentially identical to those obtained for the [1+/AdoMet] samples, showing that the cofactor binds in the same geometry to both the 1+ and 2+ states of PFL-AE. Anal. of 2D field-frequency 13C ENDOR data reveals an isotropic hyperfine contribution, which requires that AdoMet lie in contact with the cluster, weakly interacting with it through an incipient bond/antibond. From the anisotropic hyperfine contributions for the 2H and 13C ENDOR, we have estd. the distance from the closest Me proton of AdoMet to the closest iron of the cluster to be ∼3.0-3.8 Å, while the distance from the Me carbon to the nearest iron is ∼4-5 Å. We have used this information to construct a model for the interaction of AdoMet with the [4Fe-4S]2+/+ cluster of PFL-AE and have proposed a mechanism for radical generation that is consistent with these results.
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    Vey, J. L.; Drennan, C. L. Structural Insights into Radical Generation by the Radical SAM Superfamily. Chem. Rev. 2011, 111 (4), 2487– 2506,  DOI: 10.1021/cr9002616
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    Structural insights into radical generation by the radical SAM superfamily
    Vey, Jessica L.; Drennan, Catherine L.
    Chemical Reviews (Washington, DC, United States) (2011), 111 (4), 2487-2506CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. The radical adenosylmethionine (SAM) enzymes are a newly identified enzyme superfamily capable of catalyzing radical chem. similar to, but more extensive than that performed by adenosylcobalamin (AdoCbl)-dependent enzymes. The radical SAM and AdoCbl-dependent enzymes have in common the 5'-dA· intermediate, a highly oxidizing and unstable radical intermediate that has never been directly obsd. However, its existence has been shown in both the radical SAM and AdoCbl-dependent systems. Since the classification of radical SAM enzymes as a superfamily, researchers have elucidated key details of the radical generation processes, begun characterization of new radical SAM enzymes, and published the 1st few crystal structures of superfamily members. Here, the authors focus on key aspects of the 1st series of radical SAM structures in order to highlight the structural features of the superfamily and to identify the main elements involved in substrate binding and catalysis.
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    Harmer, J. E. Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions. Biochem. J. 2014, 464, 123– 133,  DOI: 10.1042/BJ20140895
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    Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions
    Harmer, Jenny E.; Hiscox, Martyn J.; Dinis, Pedro C.; Fox, Stephen J.; Iliopoulos, Andreas; Hussey, James E.; Sandy, James; Van Beek, Florian T.; Essex, Jonathan W.; Roach, Peter L.
    Biochemical Journal (2014), 464 (1), 123-133CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    Lipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) enzyme superfamily. In the present study, we solved crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX4CX5C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site-directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystd. lipoyl synthase (LipA) complex contains 5'-methylthioadenosine (MTA), a breakdown product of SAM, bound in the likely SAM-binding site. Modeling has identified an 18 Å (1 Å=0.1 nm) deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest that the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enable.
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  45. 45
    McLaughlin, M. I. Crystallographic snapshots of sulfur insertion by lipoyl synthase. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (34), 9446– 50,  DOI: 10.1073/pnas.1602486113
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    Crystallographic snapshots of sulfur insertion by lipoyl synthase
    McLaughlin, Martin I.; Lanz, Nicholas D.; Goldman, Peter J.; Lee, Kyung-Hoon; Booker, Squire J.; Drennan, Catherine L.
    Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (34), 9446-9450CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at the unactivated C6 and C8 positions of a protein-bound octanoyl chain to produce the lipoyl cofactor. To activate its substrate for sulfur insertion, LipA uses a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet) radical chem.; the remainder of the reaction mechanism, esp. the source of the sulfur, has been less clear. One controversial proposal involves the removal of sulfur from a second (auxiliary) [4Fe-4S] cluster on the enzyme, resulting in destruction of the cluster during each round of catalysis. Here, we present two high-resoln. crystal structures of LipA from Mycobacterium tuberculosis: one in its resting state and one at an intermediate state during turnover. In the resting state, an auxiliary [4Fe-4S] cluster has an unusual serine ligation to one of the irons. After reaction with an octanoyllysine-contg. 8-mer peptide substrate and 1 equiv AdoMet, conditions that allow for the first sulfur insertion but not the second insertion, the serine ligand dissocs. from the cluster, the iron ion is lost, and a sulfur atom that is still part of the cluster becomes covalently attached to C6 of the octanoyl substrate. This intermediate structure provides a clear picture of iron-sulfur cluster destruction in action, supporting the role of the auxiliary cluster as the sulfur source in the LipA reaction and describing a radical strategy for sulfur incorporation into completely unactivated substrates.
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    McCarthy, E. L. The A-type domain in Escherichia coli NfuA is required for regenerating the auxiliary [4Fe-4S] cluster in Escherichia coli lipoyl synthase. J. Biol. Chem. 2019, 294 (5), 1609– 1617,  DOI: 10.1074/jbc.RA118.006171
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    46
    The A-type domain in Escherichia coli NfuA is required for regenerating the auxiliary [4Fe-4S] cluster in Escherichia coli lipoyl synthase
    McCarthy, Erin L.; Rankin, Ananda N.; Dill, Zerick R.; Booker, Squire J.
    Journal of Biological Chemistry (2019), 294 (5), 1609-1617CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The lipoyl cofactor plays an integral role in several essential biol. processes. The last step in its de novo biosynthetic pathway, the attachment of two sulfur atoms at C6 and C8 of an n-octanoyllysyl chain, is catalyzed by lipoyl synthase (LipA), a member of the radical SAM superfamily. In addn. to the [4Fe-4S] cluster common to all radical SAM enzymes, LipA contains a second [4Fe-4S] auxiliary cluster, which is sacrificed during catalysis to supply the requisite sulfur atoms, rendering the protein inactive for further turnovers. Recently, it was shown that the Fe-S cluster carrier protein NfuA from Escherichia coli can regenerate the auxiliary cluster of E. coli LipA after each turnover, but the mol. mechanism is incompletely understood. Herein, using protein-protein interaction and kinetic assays as well as site-directed mutagenesis, we provide further insight into the mechanism of NfuA-mediated cluster regeneration. In particular, we show that the N-terminal A-type domain of E. coli NfuA is essential for its tight interaction with LipA. Further, we demonstrate that NfuA from Mycobacterium tuberculosis can also regenerate the auxiliary cluster of E. coli LipA. However, an Nfu protein from Staphylococcus aureus, which lacks the A-type domain, was severely diminished in facilitating cluster regeneration. Of note, addn. of the N-terminal domain of E. coli NfuA to S. aureus Nfu, fully restored cluster-regenerating activity. These results expand our understanding of the newly discovered mechanism by which the auxiliary cluster of LipA is restored after each turnover.
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    McCarthy, E. L.; Booker, S. J. Destruction and reformation of an iron-sulfur cluster during catalysis by lipoyl synthase. Science 2017, 358 (6361), 373– 377,  DOI: 10.1126/science.aan4574
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    Destruction and reformation of an iron-sulfur cluster during catalysis by lipoyl synthase
    McCarthy, Erin L.; Booker, Squire J.
    Science (Washington, DC, United States) (2017), 358 (6361), 373-377CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Lipoyl synthase (LipA) catalyzes the last step in the biosynthesis of the lipoyl cofactor, which is the attachment of two sulfhydryl groups to C6 and C8 of a pendant octanoyl chain. The appended sulfur atoms derive from an auxiliary [4Fe-4S] cluster on the protein that is degraded during turnover, limiting LipA to one turnover in vitro. We found that the Escherichia coli iron-sulfur (Fe-S) cluster carrier protein NfuA efficiently reconstitutes the auxiliary cluster during LipA catalysis in a step that is not rate-limiting. We also found evidence for a second pathway for cluster regeneration involving the E. coli protein IscU. These results show that enzymes that degrade their Fe-S clusters as a sulfur source can nonetheless act catalytically. Our results also explain why patients with NFU1 gene deletions exhibit phenotypes that are indicative of lipoyl cofactor deficiencies.
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    Maio, N.; Rouault, T. A. Iron-sulfur cluster biogenesis in mammalian cells: New insights into the molecular mechanisms of cluster delivery. Biochim. Biophys. Acta 2015, 1853 (6), 1493– 512,  DOI: 10.1016/j.bbamcr.2014.09.009
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    Iron -sulfur cluster biogenesis in mammalian cells: New insights into the molecular mechanisms of cluster delivery
    Maio, Nunziata; Rouault, Tracey A.
    Biochimica et Biophysica Acta, Molecular Cell Research (2015), 1853 (6), 1493-1512CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
    A review. Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorg. sulfur. The combination of the chem. reactivity of iron and sulfur, together with many variations of cluster compn., oxidn. states and protein environments, enables Fe-S clusters to participate in numerous biol. processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i.e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein-protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several crit. questions still remain, such as: what is the role of frataxin. Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload. How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer. This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of mol. features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Addnl., it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. This article is part of a Special Issue entitled: Fe/S proteins: Anal., structure, function, biogenesis and diseases.
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    Majewska, J. Binding of the Chaperone Jac1 Protein and Cysteine Desulfurase Nfs1 to the Iron-Sulfur Cluster Scaffold Isu Protein Is Mutually Exclusive. J. Biol. Chem. 2013, 288 (40), 29134– 29142,  DOI: 10.1074/jbc.M113.503524
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    Binding of the Chaperone Jac1 Protein and Cysteine Desulfurase Nfs1 to the Iron-Sulfur Cluster Scaffold Isu Protein Is Mutually Exclusive
    Majewska, Julia; Ciesielski, Szymon J.; Schilke, Brenda; Kominek, Jacek; Blenska, Anna; Delewski, Wojciech; Song, Ji-Yoon; Marszalek, Jaroslaw; Craig, Elizabeth A.; Dutkiewicz, Rafal
    Journal of Biological Chemistry (2013), 288 (40), 29134-29142CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Biogenesis of mitochondrial iron-sulfur (Fe/S) cluster proteins requires the interaction of multiple proteins with the highly conserved 14-kDa scaffold protein Isu, on which clusters are built prior to their transfer to recipient proteins. For example, the assembly process requires the cysteine desulfurase Nfs1, which serves as the sulfur donor for cluster assembly. The transfer process requires Jac1, a J-protein Hsp70 cochaperone. We recently identified three residues on the surface of Jac1 that form a hydrophobic patch crit. for interaction with Isu. The results of mol. modeling of the Isu1-Jac1 interaction, which was guided by these exptl. data and structural/biophys. information available for bacterial homologs, predicted the importance of three hydrophobic residues forming a patch on the surface of Isu1 for interaction with Jac1. Using Isu variants having alterations in residues that form the hydrophobic patch on the surface of Isu, this prediction was exptl. validated by in vitro binding assays. In addn., Nfs1 was found to require the same hydrophobic residues of Isu for binding, as does Jac1, suggesting that Jac1 and Nfs1 binding is mutually exclusive. In support of this conclusion, Jac1 and Nfs1 compete for binding to Isu. Evolutionary anal. revealed that residues involved in these interactions are conserved and that they are crit. residues for the biogenesis of Fe/S cluster protein in vivo. We propose that competition between Jac1 and Nfs1 for Isu binding plays an important role in transitioning the Fe/S cluster biogenesis machinery from the cluster assembly step to the Hsp70-mediated transfer of the Fe/S cluster to recipient proteins.
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    Vickery, L. E.; Cupp-Vickery, J. R. Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation. Crit Rev. Biochem Mol. Biol. 2007, 42 (2), 95– 111,  DOI: 10.1080/10409230701322298
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    Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation
    Vickery, Larry E.; Cupp-Vickery, Jill R.
    Critical Reviews in Biochemistry and Molecular Biology (2007), 42 (2), 95-111CODEN: CRBBEJ; ISSN:1040-9238. (Informa Healthcare USA, Inc.)
    A review. Genetic and biochem. studies have led to the identification of several cellular pathways for the biosynthesis of iron-sulfur proteins in different organisms. The most broadly distributed and highly conserved system involves an Hsp70 chaperone and J-protein co-chaperone system that interacts with a scaffold-like protein involved in [FeS]-cluster preassembly. Specialized forms of Hsp70 and their co-chaperones have evolved in bacteria (HscA, HscB) and in certain fungi (Ssq1, Jac1), whereas most eukaryotes employ a multifunctional mitochondrial Hsp70 (mtHsp70) together with a specialized co-chaperone homologous to HscB/Jac1. HscA and Ssq1 have been shown to specifically bind to a conserved sequence present in the [FeS]-scaffold protein designated IscU in bacteria and Isu in fungi, and the crystal structure of a complex of a peptide contg. the IscU recognition region bound to the HscA substrate binding domain has been detd. The interaction of IscU/Isu with HscA/Ssq1 is regulated by HscB/Jac1 which bind the scaffold protein to assist delivery to the chaperone and stabilize the chaperone-scaffold complex by enhancing chaperone ATPase activity. The crystal structure of HscB reveals that the N-terminal J-domain involved in regulation of HscA ATPase activity is similar to other J-proteins, whereas the C-terminal domain is unique and appears to mediate specific interactions with IscU. At the present time the exact function(s) of chaperone-[FeS]-scaffold interactions in iron-sulfur protein biosynthesis remain(s) to be established. In vivo and in vitro studies of yeast Ssq1 and Jac1 indicate that the chaperones are not required for [FeS]-cluster assembly on Isu. Recent in vitro studies using bacterial HscA, HscB and IscU have shown that the chaperones destabilize the IscU[FeS] complex and facilitate cluster delivery to an acceptor apo-protein consistent with a role in regulating cluster release and transfer. Addnl. genetic and biochem. studies are needed to extend these findings to mtHsp70 activities in higher eukaryotes.
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    Fox, N. G. The Human Iron-Sulfur Assembly Complex Catalyzes the Synthesis of [2Fe-2S] Clusters on ISCU2 That Can. Be Transferred to Acceptor Molecules. Biochemistry 2015, 54 (25), 3871– 9,  DOI: 10.1021/bi5014485
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    The Human Iron-Sulfur Assembly Complex Catalyzes the Synthesis of [2Fe-2S] Clusters on ISCU2 That Can Be Transferred to Acceptor Molecules
    Fox, Nicholas G.; Chakrabarti, Mrinmoy; McCormick, Sean P.; Lindahl, Paul A.; Barondeau, David P.
    Biochemistry (2015), 54 (25), 3871-3879CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Iron-sulfur (Fe-S) clusters are essential protein cofactors for most life forms. In human mitochondria, the core Fe-S biosynthetic enzymic complex (called SDUF) consists of NFS1, ISD11, ISCU2, and frataxin (FXN) protein components. Few mechanistic details about how this complex synthesizes Fe-S clusters and how these clusters are delivered to targets are known. Here CD and Mossbauer spectroscopies were used to reveal details of the Fe-S cluster assembly reaction on the SDUF complex. SDUF reactions generated [2Fe-2S] cluster intermediates that readily converted to stable [2Fe-2S] clusters bound to uncomplexed ISCU2. Similar reactions that included the apo Fe-S acceptor protein human ferredoxin (FDX1) resulted in formation of [2Fe-2S]-ISCU2 rather than [2Fe-2S]-FDX1. Subsequent addn. of dithiothreitol (DTT) induced transfer of the cluster from ISCU2 to FDX1, suggesting that [2Fe-2S]-ISCU2 is an intermediate. Reactions that initially included DTT rapidly generated [2Fe-2S]-FDX1 and bypassed formation of [2Fe-2S]-ISCU2. In the absence of apo-FDX1, incubation of [2Fe-2S]-ISCU2 with DTT generated [4Fe-4S]-ISCU2 species. Together, these results conflict with a recent report of stable [4Fe-4S] cluster formation on the SDUF complex. Rather, they support a model in which SDUF builds transient [2Fe-2S] cluster intermediates that generate clusters on sulfur-contg. mols., including uncomplexed ISCU2. Addnl. small mol. or protein factors are required for the transfer of these clusters to Fe-S acceptor proteins or the synthesis of [4Fe-4S] clusters.
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    Braymer, J. J.; Lill, R. Iron-sulfur cluster biogenesis and trafficking in mitochondria. J. Biol. Chem. 2017, 292 (31), 12754– 12763,  DOI: 10.1074/jbc.R117.787101
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    Iron-sulfur cluster biogenesis and trafficking in mitochondria
    Braymer, Joseph J.; Lill, Roland
    Journal of Biological Chemistry (2017), 292 (31), 12754-12763CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    A review. The biogenesis of Fe-S proteins in eukaryotes is a multistage, multicompartment process that is essential for a broad range of cellular functions, including genome maintenance, protein translation, energy conversion, and the antiviral response. Genetic and cell biol. studies over almost 2 decades have revealed ∼30 proteins involved in the synthesis of cellular [2Fe-2S] and [4Fe-4S] clusters and their incorporation into numerous apoproteins. Mechanistic aspects of Fe-S protein biogenesis continue to be elucidated by biochem. and ultrastructural investigations. Here, we review recent developments in the pursuit of constructing a comprehensive model of Fe-S protein assembly in the mitochondrion.
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    Braymer, J. J. Mechanistic concepts of iron-sulfur protein biogenesis in Biology. Biochim Biophys Acta Mol. Cell Res. 2021, 1868 (1), 118863,  DOI: 10.1016/j.bbamcr.2020.118863
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    Mechanistic concepts of iron-sulfur protein biogenesis in Biology
    Braymer, Joseph J.; Freibert, Sven A.; Rakwalska-Bange, Magdalena; Lill, Roland
    Biochimica et Biophysica Acta, Molecular Cell Research (2021), 1868 (1), 118863CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
    A review. Iron-sulfur (Fe/S) proteins are present in virtually all living organisms and are involved in numerous cellular processes such as respiration, photosynthesis, metabolic reactions, nitrogen fixation, radical biochem., protein synthesis, antiviral defense, and genome maintenance. Their versatile functions may go back to the proposed role of their Fe/S cofactors in the origin of life as efficient catalysts and electron carriers. In prokaryotes, three Fe/S protein biogenesis systems are known; ISC, SUF, and the more specialized NIF. The former two systems have been transferred by endosymbiosis from bacteria to mitochondria and plastids, resp., of eukaryotes. In their cytosol, eukaryotes use the CIA machinery for the biogenesis of cytosolic and nuclear Fe/S proteins. Despite the structural diversity of the protein constituents of these four machineries, general mechanistic concepts underlie the complex process of Fe/S protein biogenesis. This provides a comprehensive and comparative overview of the various known biogenesis systems in Biol., and summarizes their common or diverging mol. mechanisms, thereby illustrating both the conservation and diverse adaptions of these four machineries during evolution and under different lifestyles. Knowledge of these fundamental biochem. pathways is not only of basic scientific interest, but is important for the understanding of human 'Fe/S diseases' and can be used in biotechnol.
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    Pérard, J.; Ollagnier de Choudens, S. Iron–sulfur clusters biogenesis by the SUF machinery: close to the molecular mechanism understanding. JBIC Journal of Biological Inorganic Chemistry 2018, 23 (4), 581– 596,  DOI: 10.1007/s00775-017-1527-3
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    Iron-sulfur clusters biogenesis by the SUF machinery: close to the molecular mechanism understanding
    Perard, J.; Ollagnier de Choudens, Sandrine
    JBIC, Journal of Biological Inorganic Chemistry (2018), 23 (4), 581-596CODEN: JJBCFA; ISSN:0949-8257. (Springer)
    Review. Fe-S clusters are among the most ancient and versatile inorg. cofactors in Nature which are used by proteins for fundamental biol. processes. Multiprotein machineries (NIF, ISC, SUF) exist for Fe-S cluster biogenesis which are mainly conserved from bacteria to human. The SUF system (sufABCDSE operon) plays a general role in many bacteria under conditions of Fe limitation or oxidative stress. Here, we summarize the current understanding of the mol. mechanism of Fe-S biogenesis by the SUF machinery. The advances in our understanding of the mol. aspects of SUF originate from biochem., biophys. and recent structural studies. Combined with recent in vivo expts., the understanding of the Fe-S biogenesis mechanism has considerably moved forward.
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    Rouault, T. A.; Maio, N. Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways. J. Biol. Chem. 2017, 292 (31), 12744– 12753,  DOI: 10.1074/jbc.R117.789537
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    Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways
    Rouault, Tracey A.; Maio, Nunziata
    Journal of Biological Chemistry (2017), 292 (31), 12744-12753CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    A review. Fe-S cofactors are composed of iron and inorg. sulfur in various stoichiometries. A complex assembly pathway conducts their initial synthesis and subsequent binding to recipient proteins. In this minireview, we discuss how discovery of the role of the mammalian cytosolic aconitase, known as iron regulatory protein 1 (IRP1), led to the characterization of the function of its Fe-S cluster in sensing and regulating cellular iron homeostasis. Moreover, we present an overview of recent studies that have provided insights into the mechanism of Fe-S cluster transfer to recipient Fe-S proteins.
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    Rouault, T. A. Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease. Dis Model Mech 2012, 5 (2), 155– 64,  DOI: 10.1242/dmm.009019
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    Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease
    Rouault Tracey A
    Disease models & mechanisms (2012), 5 (2), 155-64 ISSN:.
    Iron-sulfur (Fe-S) clusters are ubiquitous cofactors composed of iron and inorganic sulfur. They are required for the function of proteins involved in a wide range of activities, including electron transport in respiratory chain complexes, regulatory sensing, photosynthesis and DNA repair. The proteins involved in the biogenesis of Fe-S clusters are evolutionarily conserved from bacteria to humans, and many insights into the process of Fe-S cluster biogenesis have come from studies of model organisms, including bacteria, fungi and plants. It is now clear that several rare and seemingly dissimilar human diseases are attributable to defects in the basic process of Fe-S cluster biogenesis. Although these diseases -which include Friedreich's ataxia (FRDA), ISCU myopathy, a rare form of sideroblastic anemia, an encephalomyopathy caused by dysfunction of respiratory chain complex I and multiple mitochondrial dysfunctions syndrome - affect different tissues, a feature common to many of them is that mitochondrial iron overload develops as a secondary consequence of a defect in Fe-S cluster biogenesis. This Commentary outlines the basic steps of Fe-S cluster biogenesis as they have been defined in model organisms. In addition, it draws attention to refinements of the process that might be specific to the subcellular compartmentalization of Fe-S cluster biogenesis proteins in some eukaryotes, including mammals. Finally, it outlines several important unresolved questions in the field that, once addressed, should offer important clues into how mitochondrial iron homeostasis is regulated, and how dysfunction in Fe-S cluster biogenesis can contribute to disease.
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    Banci, L. [2Fe-2S] cluster transfer in iron–sulfur protein biogenesis. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (17), 6203,  DOI: 10.1073/pnas.1400102111
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    [2Fe-2S] cluster transfer in iron-sulfur protein biogenesis
    Banci, Lucia; Brancaccio, Diego; Ciofi-Baffoni, Simone; Del Conte, Rebecca; Gadepalli, Ravisekhar; Mikolajczyk, Maciej; Neri, Sara; Piccioli, Mario; Winkelmann, Julia
    Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (17), 6203-6208CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Monothiol glutaredoxins play a crucial role in Fe-S protein biogenesis. Essentially all of them can coordinate a [2Fe-2S] cluster and have been proposed to mediate the transfer of [2Fe-2S] clusters from scaffold proteins to target apoproteins, possibly by acting as cluster transfer proteins. The mol. basis of [2Fe-2S] cluster transfer from monothiol glutaredoxins to target proteins is a fundamental, but still unresolved, aspect to be defined in Fe/S protein biogenesis. In mitochondria, monothiol glutaredoxin 5 (GRX5) is involved in the maturation of all cellular Fe-S proteins and participates in cellular Fe regulation. Here, the authors show that the structural plasticity of the dimeric state of the [2Fe-2S] bound form of human GRX5 (holo hGRX5) is the crucial factor that allows an efficient cluster transfer to the partner proteins human ISCA1 and ISCA2 by a specific protein-protein recognition mechanism. Holo hGRX5 works as a metallochaperone preventing the [2Fe-2S] cluster to be released in soln. in the presence of physiol. concns. of glutathione and forming a transient, cluster-mediated protein-protein intermediate with 2 physiol. protein partners receiving the [2Fe-2S] cluster. The cluster transfer mechanism defined here may extend to other mitochondrial [2Fe-2S] target proteins.
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    Nasta, V. A pathway for assembling [4Fe-4S](2+) clusters in mitochondrial iron-sulfur protein biogenesis. Febs j 2020, 287 (11), 2312– 2327,  DOI: 10.1111/febs.15140
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    A pathway for assembling [4Fe-4S]2+ clusters in mitochondrial iron-sulfur protein biogenesis
    Nasta, Veronica; Suraci, Dafne; Gourdoupis, Spyridon; Ciofi-Baffoni, Simone; Banci, Lucia
    FEBS Journal (2020), 287 (11), 2312-2327CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
    During its late steps, the mitochondrial iron-sulfur cluster (ISC) assembly machinery leads to the formation of [4Fe-4S] clusters. In vivo studies revealed that several proteins are implicated in the biosynthesis and trafficking of [4Fe-4S] clusters in mitochondria. However, they do not provide a clear picture into how these proteins cooperate. Here, we showed that three late-acting components of the mitochondrial ISC assembly machinery (GLRX5, BOLA3, and NFU1) are part of a ISC assembly pathway leading to the synthesis of a [4Fe-4S]2+ cluster on NFU1. We showed that the [2Fe-2S]2+ GLRX5-BOLA3 complex transfers its cluster to monomeric apo NFU1 to form, in the presence of a reductant, a [4Fe-4S]2+ cluster bound to dimeric NFU1. The cluster formation on NFU1 does not occur with [2Fe-2S]2+ GLRX5, and thus, the [4Fe-4S] cluster assembly pathway is activated only in the presence of BOLA3. These results define NFU1 as an 'assembler' of [4Fe-4S] clusters, i.e., a protein able of converting two [2Fe-2S]2+ clusters into a [4Fe-4S]2+ cluster. Finally, we found that the [4Fe-4S]2+ cluster bound to NFU1 has a coordination site which is easily accessible to sulfur-contg. ligands, as is typically obsd. in metallochaperones. This finding supports a role for NFU1 in promoting rapid and controlled cluster-exchange reaction.
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    Baker, P. R.; Friederich, M. W.; Swanson, M. A.; Shaikh, T.; Bhattacharya, K.; Scharer, G. H.; Aicher, J.; Creadon-Swindell, G.; Geiger, E.; MacLean, K. N. Variant non ketotic hyperglycinemia is caused by mutations in LIAS, BOLA3 and the novel gene GLRX5. Brain 2014, 137 (2), 366– 379,  DOI: 10.1093/brain/awt328
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    Variant non ketotic hyperglycinemia is caused by mutations in LIAS, BOLA3 and the novel gene GLRX5
    Baker Peter R 2nd; Friederich Marisa W; Swanson Michael A; Shaikh Tamim; Bhattacharya Kaustuv; Scharer Gunter H; Aicher Joseph; Creadon-Swindell Geralyn; Geiger Elizabeth; MacLean Kenneth N; Lee Wang-Tso; Deshpande Charu; Freckmann Mary-Louise; Shih Ling-Yu; Wasserstein Melissa; Rasmussen Malene B; Lund Allan M; Procopis Peter; Cameron Jessie M; Robinson Brian H; Brown Garry K; Brown Ruth M; Compton Alison G; Dieckmann Carol L; Collard Renata; Coughlin Curtis R 2nd; Spector Elaine; Wempe Michael F; Van Hove Johan L K
    Brain : a journal of neurology (2014), 137 (Pt 2), 366-79 ISSN:.
    Patients with nonketotic hyperglycinemia and deficient glycine cleavage enzyme activity, but without mutations in AMT, GLDC or GCSH, the genes encoding its constituent proteins, constitute a clinical group which we call 'variant nonketotic hyperglycinemia'. We hypothesize that in some patients the aetiology involves genetic mutations that result in a deficiency of the cofactor lipoate, and sequenced genes involved in lipoate synthesis and iron-sulphur cluster biogenesis. Of 11 individuals identified with variant nonketotic hyperglycinemia, we were able to determine the genetic aetiology in eight patients and delineate the clinical and biochemical phenotypes. Mutations were identified in the genes for lipoate synthase (LIAS), BolA type 3 (BOLA3), and a novel gene glutaredoxin 5 (GLRX5). Patients with GLRX5-associated variant nonketotic hyperglycinemia had normal development with childhood-onset spastic paraplegia, spinal lesion, and optic atrophy. Clinical features of BOLA3-associated variant nonketotic hyperglycinemia include severe neurodegeneration after a period of normal development. Additional features include leukodystrophy, cardiomyopathy and optic atrophy. Patients with lipoate synthase-deficient variant nonketotic hyperglycinemia varied in severity from mild static encephalopathy to Leigh disease and cortical involvement. All patients had high serum and borderline elevated cerebrospinal fluid glycine and cerebrospinal fluid:plasma glycine ratio, and deficient glycine cleavage enzyme activity. They had low pyruvate dehydrogenase enzyme activity but most did not have lactic acidosis. Patients were deficient in lipoylation of mitochondrial proteins. There were minimal and inconsistent changes in cellular iron handling, and respiratory chain activity was unaffected. Identified mutations were phylogenetically conserved, and transfection with native genes corrected the biochemical deficiency proving pathogenicity. Treatments of cells with lipoate and with mitochondrially-targeted lipoate were unsuccessful at correcting the deficiency. The recognition of variant nonketotic hyperglycinemia is important for physicians evaluating patients with abnormalities in glycine as this will affect the genetic causation and genetic counselling, and provide prognostic information on the expected phenotypic course.
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    Maio, N.; Jain, A.; Rouault, T. A. Mammalian iron-sulfur cluster biogenesis: Recent insights into the roles of frataxin, acyl carrier protein and ATPase-mediated transfer to recipient proteins. Curr. Opin Chem. Biol. 2020, 55, 34– 44,  DOI: 10.1016/j.cbpa.2019.11.014
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    Mammalian iron-sulfur cluster biogenesis: Recent insights into the roles of frataxin, acyl carrier protein and ATPase-mediated transfer to recipient proteins
    Maio, Nunziata; Jain, Anshika; Rouault, Tracey A.
    Current Opinion in Chemical Biology (2020), 55 (), 34-44CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    A review. The recently solved crystal structures of the human cysteine desulfurase NFS1, in complex with the LYR protein ISD11, the acyl carrier protein ACP, and the main scaffold ISCU, have shed light on the mol. interactions that govern initial cluster assembly on ISCU. Here, we aim to highlight recent insights into iron-sulfur (Fe-S) cluster (ISC) biogenesis in mammalian cells that have arisen from the crystal structures of the core ISC assembly complex. We will also discuss how ISCs are delivered to recipient proteins and the challenges that remain in dissecting the pathways that deliver clusters to numerous Fe-S recipient proteins in both the mitochondrial matrix and cytosolic compartments of mammalian cells.
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    Ahting, U.; Mayr, J. A.; Vanlander, A. V.; Hardy, S. A.; Santra, S.; Makowski, C.; Alston, C. L.; Zimmermann, F. A.; Abela, L.; Plecko, B. Clinical, biochemical, and genetic spectrum of seven patients with NFU1 deficiency. Front. Genet. 2015, 6, 123,  DOI: 10.3389/fgene.2015.00123
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    Clinical, biochemical, and genetic spectrum of seven patients with NFU1 deficiency
    Ahting Uwe; Mayr Johannes A; Zimmermann Franz A; Sperl Wolfgang; Vanlander Arnaud V; Smet Joel; Van Coster Rudy; Hardy Steven A; Alston Charlotte L; Taylor Robert W; Santra Saikat; Makowski Christine; Abela Lucia; Plecko Barbara; Rohrbach Marianne; Spranger Stephanie; Seneca Sara; Rolinski Boris; Hagendorff Angela; Hempel Maja; Meitinger Thomas; Prokisch Holger; Haack Tobias B; Freisinger Peter
    Frontiers in genetics (2015), 6 (), 123 ISSN:1664-8021.
    Disorders of the mitochondrial energy metabolism are clinically and genetically heterogeneous. An increasingly recognized subgroup is caused by defective mitochondrial iron-sulfur (Fe-S) cluster biosynthesis, with defects in 13 genes being linked to human disease to date. Mutations in three of them, NFU1, BOLA3, and IBA57, affect the assembly of mitochondrial [4Fe-4S] proteins leading to an impairment of diverse mitochondrial metabolic pathways and ATP production. Patients with defects in these three genes present with lactic acidosis, hyperglycinemia, and reduced activities of respiratory chain complexes I and II, the four lipoic acid-dependent 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). To date, five different NFU1 pathogenic variants have been reported in 15 patients from 12 families. We report on seven new patients from five families carrying compound heterozygous or homozygous pathogenic NFU1 mutations identified by candidate gene screening and exome sequencing. Six out of eight different disease alleles were novel and functional studies were performed to support the pathogenicity of five of them. Characteristic clinical features included fatal infantile encephalopathy and pulmonary hypertension leading to death within the first 6 months of life in six out of seven patients. Laboratory investigations revealed combined defects of pyruvate dehydrogenase complex (five out of five) and respiratory chain complexes I and II+III (four out of five) in skeletal muscle and/or cultured skin fibroblasts as well as increased lactate (five out of six) and glycine concentration (seven out of seven). Our study contributes to a better definition of the phenotypic spectrum associated with NFU1 mutations and to the diagnostic workup of future patients.
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    Cameron, J. Mutations in Iron-Sulfur Cluster Scaffold Genes NFU1 and BOLA3 Cause a Fatal Deficiency of Multiple Respiratory Chain and 2-Oxoacid Dehydrogenase Enzymes. American journal of human genetics 2011, 89, 486– 95,  DOI: 10.1016/j.ajhg.2011.08.011
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    Mutations in Iron-Sulfur Cluster Scaffold Genes NFU1 and BOLA3 Cause a Fatal Deficiency of Multiple Respiratory Chain and 2-Oxoacid Dehydrogenase Enzymes
    Cameron, Jessie M.; Janer, Alexandre; Levandovskiy, Valeriy; MacKay, Nevena; Rouault, Tracey A.; Tong, Wing-Hang; Ogilvie, Isla; Shoubridge, Eric A.; Robinson, Brian H.
    American Journal of Human Genetics (2011), 89 (4), 486-495CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
    Severe combined deficiency of the 2-oxoacid dehydrogenases, assocd. with a defect in lipoate synthesis and accompanied by defects in complexes I, II, and III of the mitochondrial respiratory chain, is a rare autosomal recessive syndrome with no obvious causative gene defect. A candidate locus for this syndrome was mapped to chromosomal region 2p14 by microcell-mediated chromosome transfer in two unrelated families. Unexpectedly, anal. of genes in this area identified mutations in two different genes, both of which are involved in [Fe-S] cluster biogenesis. A homozygous missense mutation, c.545G>A, near the splice donor of exon 6 in NFU1 predicting a p.Arg182Gln substitution was found in one of the families. The mutation results in abnormal mRNA splicing of exon 6, and no mature protein could be detected in fibroblast mitochondria. A single base-pair duplication c.123dupA was identified in BOLA3 in the second family, causing a frame shift that produces a premature stop codon (p.Glu42Argfs*13). Transduction of fibroblast lines with retroviral vectors expressing the mitochondrial, but not the cytosolic isoform of NFU1 and with isoform 1, but not isoform 2 of BOLA3 restored both respiratory chain function and oxoacid dehydrogenase complexes. NFU1 was previously proposed to be an alternative scaffold to ISCU for the biogenesis of [Fe-S] centers in mitochondria, and the function of BOLA3 was previously unknown. Our results demonstrate that both play essential roles in the prodn. of [Fe-S] centers for the normal maturation of lipoate-contg. 2-oxoacid dehydrogenases, and for the assembly of the respiratory chain complexes.
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    Navarro-Sastre, A. A fatal mitochondrial disease is associated with defective NFU1 function in the maturation of a subset of mitochondrial Fe-S proteins. Am. J. Hum. Genet. 2011, 89 (5), 656– 67,  DOI: 10.1016/j.ajhg.2011.10.005
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    A Fatal Mitochondrial Disease Is Associated with Defective NFU1 Function in the Maturation of a Subset of Mitochondrial Fe-S Proteins
    Navarro-Sastre, Aleix; Tort, Frederic; Stehling, Oliver; Uzarska, Marta A.; Arranz, Jose Antonio; del Toro, Mireia; Labayru, M. Teresa; Landa, Joseba; Font, Aida; Garcia-Villoria, Judit; Merinero, Begona; Ugarte, Magdalena; Gutierrez-Solana, Luis Gonzalez; Campistol, Jaume; Garcia-Cazorla, Angels; Vaquerizo, Julian; Riudor, Encarnacio; Briones, Paz; Elpeleg, Orly; Ribes, Antonia; Lill, Roland
    American Journal of Human Genetics (2011), 89 (5), 656-667CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
    We report on ten individuals with a fatal infantile encephalopathy and/or pulmonary hypertension, leading to death before the age of 15 mo. Hyperglycinemia and lactic acidosis were common findings. Glycine cleavage system and pyruvate dehydrogenase complex (PDHC) activities were low. Homozygosity mapping revealed a perfectly overlapping homozygous region of 1.24 Mb corresponding to chromosome 2 and led to the identification of a homozygous missense mutation (c.622G>T) in NFU1, which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. Nine individuals were homozygous for this mutation, whereas one was compd. heterozygous for this and a splice-site (c.545+5G>A) mutation. The biochem. phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS). Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases. Upon depletion of NFU1 by RNA interference in human cell culture, LAS and, in turn, PDHC activities were largely diminished. In addn., the amt. of succinate dehydrogenase, but no other Fe-S proteins, was decreased. In contrast, depletion of the general Fe-S scaffold protein ISCU severely affected assembly of all tested Fe-S proteins, suggesting that NFU1 performs a specific function in mitochondrial Fe-S cluster maturation. Similar biochem. effects were obsd. in Saccharomyces cerevisiae upon deletion of NFU1, resulting in lower lipoylation and SDH activity. Importantly, yeast Nfu1 protein carrying the individuals' missense mutation was functionally impaired. We conclude that NFU1 functions as a late-acting maturation factor for a subset of mitochondrial Fe-S proteins.
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    Wachnowsky, C. Understanding the Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1)-Impact of a Disease-Causing Gly208Cys Substitution on Structure and Activity of NFU1 in the Fe/S Cluster Biosynthetic Pathway. J. Mol. Biol. 2017, 429 (6), 790– 807,  DOI: 10.1016/j.jmb.2017.01.021
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    64
    Understanding the molecular basis of multiple mitochondrial dysfunctions syndrome 1 (MMDS1)-Impact of a disease-causing Gly208Cys substitution on structure and activity of NFU1 in the Fe/S Cluster Biosynthetic Pathway
    Wachnowsky, Christine; Wesley, Nathaniel A.; Fidai, Insiya; Cowan, J. A.
    Journal of Molecular Biology (2017), 429 (6), 790-807CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Iron-sulfur (Fe/S)-cluster-contg. proteins constitute one of the largest protein classes, with varied functions that include electron transport, regulation of gene expression, substrate binding and activation, and radical generation. Consequently, the biosynthetic machinery for Fe/S clusters is evolutionarily conserved, and mutations in a variety of putative intermediate Fe/S cluster scaffold proteins can cause disease states, including multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalomyopathy. Herein, we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (Gly208Cys) near the active site of NFU1, an Fe/S scaffold protein, via an in vitro investigation into the structural and functional consequences. Anal. of protein stability and oligomeric state demonstrates that the mutant increases the propensity to dimerize and perturbs the secondary structure compn. These changes appear to underlie the severely decreased ability of mutant NFU1 to accept an Fe/S cluster from physiol. relevant sources. Therefore, the point mutation on NFU1 impairs downstream cluster trafficking and results in the disease phenotype, because there does not appear to be an alternative in vivo reconstitution path, most likely due to greater protein oligomerization from a minor structural change.
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  65. 65
    Melber, A.; Na, U.; Vashisht, A.; Weiler, B. D; Lill, R.; Wohlschlegel, J. A; Winge, D. R Role of Nfu1 and Bol3 in iron-sulfur cluster transfer to mitochondrial clients. Elife 2016, 5, e15991,  DOI: 10.7554/eLife.15991
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    65
    Role of Nfu1 and Bol3 in iron-sulfur cluster transfer to mitochondrial clients
    Melber, Andrew; Na, Un; Vashisht, Ajay; Weiler, Benjamin D.; Lill, Roland; Wohlschlege, James A.; Winge, Dennis R.
    eLife (2016), 5 (), e15991/1-e15991/24CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
    Iron-sulfur (Fe-S) clusters are essential for many cellular processes, ranging from aerobic respiration, metabolite biosynthesis, ribosome assembly and DNA repair. Mutations in NFU1 and BOLA3 have been linked to genetic diseases with defects in mitochondrial Fe-S centers. Through genetic studies in yeast, we demonstrate that Nfu1 functions in a late step of [4Fe-4S] cluster biogenesis that is of heightened importance during oxidative metab. Proteomic studies revealed Nfu1 phys. interacts with components of the ISA [4Fe-4S] assembly complex and client proteins that need [4Fe-4S] clusters to function. Addnl. studies focused on the mitochondrial BolA proteins, Bol1 and Bol3 (yeast homolog to human BOLA3), revealing that Bol1 functions earlier in Fe-S biogenesis with the monothiol glutaredoxin, Grx5, and Bol3 functions late with Nfu1. Given these observations, we propose that Nfu1, assisted by Bol3, functions to facilitate Fe-S transfer from the biosynthetic app. to the client proteins preventing oxidative damage to [4Fe-4S] clusters.
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  66. 66
    Zhao, S. Assembly of the covalent linkage between lipoic acid and its cognate enzymes. Chem. Biol. 2003, 10, 1293– 1302,  DOI: 10.1016/j.chembiol.2003.11.016
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    Assembly of the Covalent Linkage between Lipoic Acid and Its Cognate Enzymes
    Zhao, Xin; Miller, J. Richard; Jiang, Yanfang; Marletta, Michael A.; Cronan, John E.
    Chemistry & Biology (2003), 10 (12), 1293-1302CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
    Lipoic acid is synthesized from octanoic acid by insertion of sulfur atoms at carbons 6 and 8 and is covalently attached to a pyruvate dehydrogenase (PDH) subunit. We show that sulfur atoms can be inserted into octanoyl moieties attached to a PDH subunit or a derived domain. Escherichia coli lipB mutants grew well when supplemented with octanoate in place of lipoate. Octanoate growth required both lipoate protein ligase (LplA) and LipA, the sulfur insertion protein, suggesting that LplA attached octanoate to the dehydrogenase and LipA then converted the octanoate to lipoate. This pathway was tested by labeling a PDH domain with deuterated octanoate in an E. coli strain devoid of LipA activity. The labeled octanoyl domain was converted to lipoylated domain upon restoration of LipA. Moreover, octanoyl domain and octanoyl-PDH were substrates for sulfur insertion in vitro.
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    Billgren, E. S.; Cicchillo, R. M.; Nesbitt, N. M.; Booker, S. J. Lipoic acid biosynthesis and enzymology. In Comprehensive Natural Products II Chemistry and Biology; Mander, L., Liu, H.-W., Eds.; Elsevier: Oxford, U.K., 2010; pp 181– 212.
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    Cicchillo, R. M.; Booker, S. J. Mechanistic investigations of lipoic acid biosynthesis in Escherichia coli: both sulfur atoms in lipoic acid are contributed by the same lipoyl synthase polypeptide. J. Am. Chem. Soc. 2005, 127, 2860– 2861,  DOI: 10.1021/ja042428u
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    Mechanistic Investigations of Lipoic Acid Biosynthesis in Escherichia coli: Both Sulfur Atoms in Lipoic Acid are Contributed by the Same Lipoyl Synthase Polypeptide
    Cicchillo, Robert M.; Booker, Squire J.
    Journal of the American Chemical Society (2005), 127 (9), 2860-2861CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Lipoyl synthase catalyzes the final step in the de novo biosynthesis of the lipoyl cofactor, which is the insertion of two sulfur atoms into an octanoyl chain that is bound in an amide linkage to a conserved lysine on a lipoyl-accepting protein. We show herein that the sulfur atoms in the lipoyl cofactor are derived from lipoyl synthase itself, and that each lipoyl synthase polypeptide contributes both of the sulfur atoms to the intact cofactor.
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    Cicchillo, R. M. Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid. Biochemistry 2004, 43, 6378– 6386,  DOI: 10.1021/bi049528x
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    Lipoyl Synthase Requires Two Equivalents of S-Adenosyl-L-methionine To Synthesize One Equivalent of Lipoic Acid
    Cicchillo, Robert M.; Iwig, David F.; Jones, A. Daniel; Nesbitt, Natasha M.; Baleanu-Gogonea, Camelia; Souder, Matthew G.; Tu, Loretta; Booker, Squire J.
    Biochemistry (2004), 43 (21), 6378-6386CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various α-keto acids, as well as the cleavage of glycine into CO2 and NH3, with concomitant transfer of its α-carbon to tetrahydrofolate, generating N5,N10-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochem. studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-L-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA•), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming L-methionine in addn. to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated deriv. of the lipoyl-bearing subunit (E2) of the pyruvate dehydrogenase complex. Herein, the authors show that the octanoylated deriv. of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, the authors show that the 5'-dA• acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d15]H-protein to 5'-deoxyadenosine. Last, the authors' data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.
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    Douglas, P. Lipoyl synthase inserts sulfur atoms into an octanoyl substrate in a stepwise manner. Angew. Chem. 2006, 118, 5321– 5323,  DOI: 10.1002/ange.200601910
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    McLaughlin, M. I. Crystallographic snapshots of sulfur insertion by lipoyl synthase. Proc. Natl. Acad. Sci. U S A 2016, 113, 9446– 9450,  DOI: 10.1073/pnas.1602486113
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    Crystallographic snapshots of sulfur insertion by lipoyl synthase
    McLaughlin, Martin I.; Lanz, Nicholas D.; Goldman, Peter J.; Lee, Kyung-Hoon; Booker, Squire J.; Drennan, Catherine L.
    Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (34), 9446-9450CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at the unactivated C6 and C8 positions of a protein-bound octanoyl chain to produce the lipoyl cofactor. To activate its substrate for sulfur insertion, LipA uses a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet) radical chem.; the remainder of the reaction mechanism, esp. the source of the sulfur, has been less clear. One controversial proposal involves the removal of sulfur from a second (auxiliary) [4Fe-4S] cluster on the enzyme, resulting in destruction of the cluster during each round of catalysis. Here, we present two high-resoln. crystal structures of LipA from Mycobacterium tuberculosis: one in its resting state and one at an intermediate state during turnover. In the resting state, an auxiliary [4Fe-4S] cluster has an unusual serine ligation to one of the irons. After reaction with an octanoyllysine-contg. 8-mer peptide substrate and 1 equiv AdoMet, conditions that allow for the first sulfur insertion but not the second insertion, the serine ligand dissocs. from the cluster, the iron ion is lost, and a sulfur atom that is still part of the cluster becomes covalently attached to C6 of the octanoyl substrate. This intermediate structure provides a clear picture of iron-sulfur cluster destruction in action, supporting the role of the auxiliary cluster as the sulfur source in the LipA reaction and describing a radical strategy for sulfur incorporation into completely unactivated substrates.
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    Lanz, N. D. Characterization of Lipoyl Synthase from Mycobacterium tuberculosis. Biochemistry 2016, 55 (9), 1372– 83,  DOI: 10.1021/acs.biochem.5b01216
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    Characterization of Lipoyl Synthase from Mycobacterium tuberculosis
    Lanz, Nicholas D.; Lee, Kyung-Hoon; Horstmann, Abigail K.; Pandelia, Maria-Eirini; Cicchillo, Robert M.; Krebs, Carsten; Booker, Squire J.
    Biochemistry (2016), 55 (9), 1372-1383CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The prevalence of multiple and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is on the rise, necessitating the identification of new targets to combat an organism that has infected one-third of the world's population, according to the World Health Organization. The biosynthesis of the lipoyl cofactor is one possible target, given its crit. importance in cellular metab. and the apparent lack of functional salvage pathways in Mtb that are found in humans and many other organisms. The lipoyl cofactor is synthesized de novo in two committed steps, involving the LipB-catalyzed transfer of an octanoyl chain derived from fatty acid biosynthesis to a lipoyl carrier protein and the LipA-catalyzed insertion of sulfur atoms at C6 and C8 of the octanoyl chain. A no. of in vitro studies of lipoyl synthases from Escherichia coli, Sulfolobus solfataricus, and Thermosynechococcus elongatus have been conducted, but the enzyme from Mtb has not been characterized. Herein, we show that LipA from Mtb contains two [4Fe-4S] clusters and converts an octanoyl peptide substrate to the corresponding lipoyl peptide product via the same C6-monothiolated intermediate as that obsd. in the E. coli LipA reaction. In addn., we show that LipA from Mtb forms a complex with the H protein of the glycine cleavage system and that the strength of assocn. is dependent on the presence of S-adenosyl-L-methionine. We also show that LipA from Mtb can complement a lipA mutant of E. coli, demonstrating the commonalities of the two enzymes. Lastly, we show that the substrate for LipA, which normally acts on a post-translationally modified protein, can be reduced to carboxybenzyl-octanoyllysine.
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    Camponeschi, F. Paramagnetic (1)H NMR Spectroscopy to Investigate the Catalytic Mechanism of Radical S-Adenosylmethionine Enzymes. J. Mol. Biol. 2019, 431 (22), 4514– 4522,  DOI: 10.1016/j.jmb.2019.08.018
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    73
    Paramagnetic 1H NMR Spectroscopy to Investigate the Catalytic Mechanism of Radical S-Adenosylmethionine Enzymes
    Camponeschi, Francesca; Muzzioli, Riccardo; Ciofi-Baffoni, Simone; Piccioli, Mario; Banci, Lucia
    Journal of Molecular Biology (2019), 431 (22), 4514-4522CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Iron-sulfur clusters in radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chem. challenging reactions across all domains of life. Here we showed that 1H NMR spectroscopy expts. tailored to reveal hyperfine-shifted signals of metal-ligands is a powerful tool to monitor the binding of SAM and of the octanoyl-peptide substrate to the two [4Fe-4S] clusters of human lipoyl synthase. The paramagnetically shifted signals of the iron-ligands were specifically assigned to each of the two bound [4Fe-4S] clusters, and then used to examine the interaction of SAM and substrate mols. with each of the two [4Fe-4S] clusters of human lipoyl synthase. 1H NMR spectroscopy can therefore contribute to the description of the catalityc mechanism of radical SAM enzymes.
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    Hendricks, A. L. Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly. Int. J. Mol. Sci. 2021, 22 (4), 1598,  DOI: 10.3390/ijms22041598
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    Characterization and reconstitution of human lipoyl synthase (LIAS) supports ISCA2 and ISCU as primary cluster donors and an ordered mechanism of cluster assembly
    Hendricks, Amber L.; Wachnowsky, Christine; Fries, Brian; Fidai, Insiya; Cowan, James A.
    International Journal of Molecular Sciences (2021), 22 (4), 1598CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    Lipoyl synthase (LIAS) is an iron-sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe-4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, resp. Most information concerning LIAS and its mechanism has been detd. from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron-sulfur (Fe-S) cluster reconstitution mechanisms. Cluster donation by a no. of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe-2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liq. chromatog.-mass spectrometry (LC-MS) assay. ESR (EPR) studies of native LIAS and substituted derivs. that lacked the ability to bind one or the other of LIAS's two [4Fe-4S] clusters revealed a likely order of cluster addn., with the auxiliary cluster preceding the reducing [4Fe-4S] center. These results detail the trafficking of Fe-S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe-S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe-4S] cluster reconstitution is evident.
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    Jain, A. Assembly of the [4Fe-4S] cluster of NFU1 requires the coordinated donation of two [2Fe-2S] clusters from the scaffold proteins, ISCU2 and ISCA1. Hum. Mol. Genet. 2020, 29 (19), 3165– 3182,  DOI: 10.1093/hmg/ddaa172
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    Assembly of the [4Fe-4S] cluster of NFU1 requires the coordinated donation of two [2Fe-2S] clusters from the scaffold proteins, ISCU2 and ISCA1
    Jain, Anshika; Singh, Anamika; Maio, Nunziata; Rouault, Tracey A.
    Human Molecular Genetics (2020), 29 (19), 3165-3182CODEN: HMGEE5; ISSN:1460-2083. (Oxford University Press)
    NFU1, a late-acting iron-sulfur (Fe-S) cluster carrier protein, has a key role in the pathogenesis of the disease, multiple mitochondrial dysfunctions syndrome. In this work, using genetic and biochem. approaches, we identified the initial scaffold protein, mitochondrial ISCU (ISCU2) and the secondary carrier, ISCA1, as the direct donors of Fe-S clusters to mitochondrial NFU1, which appears to dimerize and reductively mediate the formation of a bridging [4Fe-4S] cluster, aided by ferredoxin 2. By monitoring the abundance of target proteins that acquire their Fe-S clusters from NFU1, we characterized the effects of several novel pathogenic NFU1 mutations. We obsd. that NFU1 directly interacts with each of the Fe-S cluster scaffold proteins known to ligate [2Fe-2S] clusters, ISCU2 and ISCA1, and we mapped the site of interaction to a conserved hydrophobic patch of residues situated at the end of the C-terminal alpha-helix of NFU1. Furthermore, we showed that NFU1 lost its ability to acquire its Fe-S cluster when mutagenized at the identified site of interaction with ISCU2 and ISCA1, which thereby adversely affected biochem. functions of proteins that are thought to acquire their Fe-S clusters directly from NFU1, such as lipoic acid synthase, which supports the Fe-S-dependent process of lipoylation of components of multiple key enzyme complexes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and the glycine cleavage complex.
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    Johnson, D. C.; Unciuleac, M.-C.; Dean, D. R. Controlled Expression and Functional Analysis of Iron-Sulfur Cluster Biosynthetic Components within Azotobacter vinelandii. J. Bacteriol. 2006, 188 (21), 7551– 7561,  DOI: 10.1128/JB.00596-06
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    Controlled expression and functional analysis of iron-sulfur cluster biosynthetic components within Azotobacter vinelandii
    Johnson, Deborah C.; Unciuleac, Mihaela-Carmen; Dean, Dennis R.
    Journal of Bacteriology (2006), 188 (21), 7551-7561CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    A system for the controlled expression of genes in A. vinelandii by using genomic fusions to the sucrose catabolic regulon was developed. This system was used for the functional anal. of the A. vinelandii isc genes, whose products are involved in the maturation of [Fe-S] proteins. For this anal., the scrX gene, contained within the sucrose catabolic regulon, was replaced by the contiguous A. vinelandii iscS, iscU, iscA, hscB, hscA, fdx, and iscX genes, resulting in duplicate genomic copies of these genes: one whose expression is directed by the normal isc regulatory elements (Pisc) and the other whose expression is directed by the scrX promoter (PscrX). Functional anal. of [Fe-S] protein maturation components was achieved by placing a mutation within a particular Pisc-controlled gene with subsequent repression of the corresponding PscrX-controlled component by growth on glucose as the carbon source. This exptl. strategy was used to show that IscS, IscU, HscBA, and Fdx are essential in A. vinelandii and that their depletion results in a deficiency in the maturation of aconitase, an enzyme that requires a [4Fe-4S] cluster for its catalytic activity. Depletion of IscA results in a null growth phenotype only when cells are cultured under conditions of elevated oxygen, marking the 1st null phenotype assocd. with the loss of a bacterial IscA-type protein. Furthermore, the null growth phenotype of cells depleted of HscBA could be partially reversed by culturing cells under conditions of low O2. Conserved amino acid residues within IscS, IscU, and IscA that are essential for their resp. functions and/or whose replacement results in a partial or complete dominant-neg. growth phenotype were also identified using this system.
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    Pandelia, M. E. Mössbauer spectroscopy of Fe/S proteins. Biochim. Biophys. Acta - Molecular Cell Research 2015, 1853 (6), 1395– 1405,  DOI: 10.1016/j.bbamcr.2014.12.005
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    M.ovrddot.ossbauer spectroscopy of Fe/S proteins
    Pandelia, Maria-Eirini; Lanz, Nicholas D.; Booker, Squire J.; Krebs, Carsten
    Biochimica et Biophysica Acta, Molecular Cell Research (2015), 1853 (6), 1395-1405CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
    A review. Iron-sulfur (Fe/S) clusters are structurally and functionally diverse cofactors that are found in all domains of life. 57Fe Mossbauer spectroscopy is a technique that provides information about the chem. nature of all chem. distinct Fe species contained in a sample, such as Fe oxidn. and spin state, nuclearity of a cluster with more than one metal ion, electron spin ground state of the cluster, and delocalization properties in mixed-valent clusters. Moreover, the technique allows for quantitation of all Fe species, when it is used in conjunction with ESR (EPR) spectroscopy and anal. methods. 57Fe-Mossbauer spectroscopy played a pivotal role in unraveling the electronic structures of the "well-established" [2Fe-2S]2+/+, [3Fe-4S]1+/0, and [4Fe-4S]3+/2+/1+/0 clusters and -more-recently- was used to characterize novel Fe/S clustsers, including the [4Fe-3S] cluster of the O2-tolerant hydrogenase from Aquifex aeolicus and the 3Fe-cluster intermediate obsd. during the reaction of lipoyl synthase, a member of the radical SAM enzyme superfamily.
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    Cai, K. Structural/Functional Properties of Human NFU1, an Intermediate [4Fe-4S] Carrier in Human Mitochondrial Iron-Sulfur Cluster Biogenesis. Structure 2016, 24 (12), 2080– 2091,  DOI: 10.1016/j.str.2016.08.020
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    Structural/Functional Properties of Human NFU1, an Intermediate [4Fe-4S] Carrier in Human Mitochondrial Iron-Sulfur Cluster Biogenesis
    Cai, Kai; Liu, Gaohua; Frederick, Ronnie O.; Xiao, Rong; Montelione, Gaetano T.; Markley, John L.
    Structure (Oxford, United Kingdom) (2016), 24 (12), 2080-2091CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
    Human mitochondrial NFU1 functions in the maturation of iron-sulfur proteins, and NFU1 deficiency is assocd. with a fatal mitochondrial disease. We detd. three-dimensional structures of the N- and C-terminal domains of human NFU1 by NMR spectroscopy and used these structures along with small-angle X-ray scattering (SAXS) data to derive structural models for full-length monomeric apo-NFU1, dimeric apo-NFU1 (an artifact of intermol. disulfide bond formation), and holo-NFUI (the [4Fe-4S] cluster-contg. form of the protein). Apo-NFU1 contains two cysteine residues in its C-terminal domain, and two apo-NFU1 subunits coordinate one [4Fe-4S] cluster to form a cluster-linked dimer. Holo-NFU1 consists of a complex of three of these dimers as shown by mol. wt. ests. from SAXS and size-exclusion chromatog. The SAXS-derived structural model indicates that one N-terminal region from each of the three dimers forms a tripartite interface. The activity of the holo-NFU1 prepn. was verified by demonstrating its ability to activate apo-aconitase.
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    Wachnowsky, C. Regulation of human Nfu activity in Fe-S cluster delivery-characterization of the interaction between Nfu and the HSPA9/Hsc20 chaperone complex. Febs j 2018, 285 (2), 391– 410,  DOI: 10.1111/febs.14353
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    Regulation of human Nfu activity in Fe-S cluster delivery - characterization of the interaction between Nfu and the HSPA9/Hsc20 chaperone complex
    Wachnowsky, Christine; Liu, Yushi; Yoon, Taejin; Cowan, J. A.
    FEBS Journal (2018), 285 (2), 391-410CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
    Iron-sulfur cluster biogenesis is a complex, but highly regulated process that involves de novo cluster formation from iron and sulfide ions on a scaffold protein, and subsequent delivery to final targets via a series of Fe-S cluster-binding carrier proteins. The process of cluster release from the scaffold/carrier for transfer to the target proteins may be mediated by a dedicated Fe-S cluster chaperone system. In human cells, the chaperones include heat shock protein HSPA9 and the J-type chaperone Hsc20. While the role of chaperones has been somewhat clarified in yeast and bacterial systems, many questions remain over their functional roles in cluster delivery and interactions with a variety of human Fe-S cluster proteins. One such protein, Nfu, has recently been recognized as a potential interaction partner of the chaperone complex. Herein, we examd. the ability of human Nfu to function as a carrier by interacting with the human chaperone complex. Human Nfu is shown to bind to both chaperone proteins with binding affinities similar to those obsd. for IscU binding to the homologous HSPA9 and Hsc20, while Nfu can also stimulate the ATPase activity of HSPA9. Addnl., the chaperone complex was able to promote Nfu function by enhancing the second-order rate consts. for Fe-S cluster transfer to target proteins and providing directionality in cluster transfer from Nfu by eliminating promiscuous transfer reactions. Together, these data support a hypothesis in which Nfu can serve as an alternative carrier protein for chaperone-mediated cluster release and delivery in Fe-S cluster biogenesis and trafficking.
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  80. 80
    Wesley, N. A.; Wachnowsky, C.; Fidai, I.; Cowan, J. A. Understanding the molecular basis for multiple mitochondrial dysfunctions syndrome 1 (MMDS1): impact of a disease-causing Gly189Arg substitution on NFU1. FEBS J. 2017, 284, 3838– 3848,  DOI: 10.1111/febs.14271
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    80
    Understanding the molecular basis for multiple mitochondrial dysfunctions syndrome 1 (MMDS1): impact of a disease-causing Gly189Arg substitution on NFU1
    Wesley, Nathaniel A.; Wachnowsky, Christine; Fidai, Insiya; Cowan, J. A.
    FEBS Journal (2017), 284 (22), 3838-3848CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
    Iron-sulfur (Fe/S) cluster-contg. proteins constitute one of the largest protein classes, with highly varied function. Consequently, the biosynthesis of Fe/S clusters is evolutionarily conserved and mutations in intermediate Fe/S cluster scaffold proteins can cause disease, including multiple mitochondrial dysfunctions syndrome (MMDS). Herein, we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (p.Gly189Arg) near the active site of NFU1, an Fe/S scaffold protein. In vitro investigation into the structure-function relationship of the Gly189Arg deriv., along with two other variants, reveals that substitution at position 189 triggers structural changes that increase flexibility, decrease stability, and alter the monomer-dimer equil. toward monomer, thereby impairing the ability of the Gly189X derivs. to receive an Fe/S cluster from physiol. relevant sources.
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    Jumper, J. Highly accurate protein structure prediction with AlphaFold. Nature 2021, 596 (7873), 583– 589,  DOI: 10.1038/s41586-021-03819-2
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    Highly accurate protein structure prediction with AlphaFold
    Jumper, John; Evans, Richard; Pritzel, Alexander; Green, Tim; Figurnov, Michael; Ronneberger, Olaf; Tunyasuvunakool, Kathryn; Bates, Russ; Zidek, Augustin; Potapenko, Anna; Bridgland, Alex; Meyer, Clemens; Kohl, Simon A. A.; Ballard, Andrew J.; Cowie, Andrew; Romera-Paredes, Bernardino; Nikolov, Stanislav; Jain, Rishub; Adler, Jonas; Back, Trevor; Petersen, Stig; Reiman, David; Clancy, Ellen; Zielinski, Michal; Steinegger, Martin; Pacholska, Michalina; Berghammer, Tamas; Bodenstein, Sebastian; Silver, David; Vinyals, Oriol; Senior, Andrew W.; Kavukcuoglu, Koray; Kohli, Pushmeet; Hassabis, Demis
    Nature (London, United Kingdom) (2021), 596 (7873), 583-589CODEN: NATUAS; ISSN:0028-0836. (Nature Portfolio)
    Proteins are essential to life, and understanding their structure can facilitate a mechanistic understanding of their function. Through an enormous exptl. effort, the structures of around 100,000 unique proteins have been detd., but this represents a small fraction of the billions of known protein sequences. Structural coverage is bottlenecked by the months to years of painstaking effort required to det. a single protein structure. Accurate computational approaches are needed to address this gap and to enable large-scale structural bioinformatics. Predicting the three-dimensional structure that a protein will adopt based solely on its amino acid sequence-the structure prediction component of the 'protein folding problem'-has been an important open research problem for more than 50 years. Despite recent progress, existing methods fall far short of at. accuracy, esp. when no homologous structure is available. Here we provide the first computational method that can regularly predict protein structures with at. accuracy even in cases in which no similar structure is known. We validated an entirely redesigned version of our neural network-based model, AlphaFold, in the challenging 14th Crit. Assessment of protein Structure Prediction (CASP14), demonstrating accuracy competitive with exptl. structures in a majority of cases and greatly outperforming other methods. Underpinning the latest version of AlphaFold is a novel machine learning approach that incorporates phys. and biol. knowledge about protein structure, leveraging multi-sequence alignments, into the design of the deep learning algorithm.
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    Cai, K.; Frederick, R. O.; Markley, J. L. ISCU interacts with NFU1, and ISCU[4Fe-4S] transfers its Fe-S cluster to NFU1 leading to the production of holo-NFU1. J. Struct Biol. 2020, 210 (2), 107491,  DOI: 10.1016/j.jsb.2020.107491
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    82
    ISCU interacts with NFU1, and ISCU[4Fe-4S] transfers its Fe-S cluster to NFU1 leading to the production of holo-NFU1
    Cai, Kai; Frederick, Ronnie O.; Markley, John L.
    Journal of Structural Biology (2020), 210 (2), 107491CODEN: JSBIEM; ISSN:1047-8477. (Elsevier Inc.)
    NFU1 is a late-acting factor in the biogenesis of human mitochondrial iron-sulfur proteins. Mutations in NFU1 are assocd. with genetic diseases such as multiple mitochondrial dysfunctions syndrome 1 (MMDS1) that involve defects in mitochondrial [4Fe-4S] proteins. We present results from NMR spectroscopy, small angle X-ray scattering, size exclusion chromatog., and isothermal titrn. calorimetry showing that the structured conformer of human ISCU binds human NFU1. The dissocn. const. detd. by ITC is Kd = 1.1 ± 0.2 μM. NMR and SAXS studies led to a structural model for the complex in which the cluster binding region of ISCU interacts with two α-helixes in the C-terminal domain of NFU1. In vitro expts. demonstrate that ISCU[4Fe-4S] transfers its Fe-S cluster to apo-NFU1, in the absence of a chaperone, leading to the assembly of holo-NFU1. By contrast, the cluster of ISCU[2Fe-2S] remains bound to ISCU in the presence of apo-NFU1.
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  83. 83
    Uzarska, M. A; Nasta, V.; Weiler, B. D; Spantgar, F.; Ciofi-Baffoni, S.; Saviello, M. R.; Gonnelli, L.; Muhlenhoff, U.; Banci, L.; Lill, R. Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins. Elife 2016, 5, e16673,  DOI: 10.7554/eLife.16673
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    84
    Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins
    Uzarska, Marta A.; Nasta, Veronica; Weiler, Benjamin D.; Spantgar, Farah; CiofiBaffoni, Simone; Saviello, Maria Rosaria; Gonnelli, Leonardo; Muhlenhoff, Ulrich; Banci, Lucia; Lill, Roland
    eLife (2016), 5 (), e16673/1-e16673/25, S1-S8CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
    Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochem. basis for explaining the pathol. phenotypes of patients with mutations in BOLA3.
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    Sheftel, A. D. The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation. Mol. Biol. Cell 2012, 23 (7), 1157– 66,  DOI: 10.1091/mbc.e11-09-0772
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    85
    The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation
    Sheftel, Alex D.; Wilbrecht, Claudia; Stehling, Oliver; Niggemeyer, Brigitte; Elsaesser, Hans-Peter; Muehlenhoff, Ulrich; Lill, Roland
    Molecular Biology of the Cell (2012), 23 (7), 1157-1166CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
    Members of the bacterial and mitochondrial iron-sulfur cluster (ISC) assembly machinery include the so-called A-type ISC proteins, which support the assembly of a subset of Fe/S apoproteins. The human genome encodes two A-type proteins, termed ISCA1 and ISCA2, which are related to Saccharomyces cerevisiae Isa1 and Isa2, resp. An addnl. protein, Iba57, phys. interacts with Isa1 and Isa2 in yeast. To test the cellular role of human ISCA1, ISCA2, and IBA57, HeLa cells were depleted for any of these proteins by RNA interference technol. Depleted cells contained massively swollen and enlarged mitochondria that were virtually devoid of cristae membranes, demonstrating the importance of these proteins for mitochondrial biogenesis. The activities of mitochondrial [4Fe-4S] proteins, including aconitase, respiratory complex I, and lipoic acid synthase, were diminished following depletion of the three proteins. In contrast, the mitochondrial [2Fe-2S] enzyme ferrochelatase and cellular heme content were unaffected. We further provide evidence against a localization and direct Fe/S protein maturation function of ISCA1 and ISCA2 in the cytosol. Taken together, our data suggest that ISCA1, ISCA2, and IBA57 are specifically involved in the maturation of mitochondrial [4Fe-4S] proteins functioning late in the ISC assembly pathway.
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    Beinert, H.; Holm, R. H.; Münck, E. Iron-sulfur clusters: nature’s modular, multipurpose structures. Science 1997, 277, 653– 659,  DOI: 10.1126/science.277.5326.653
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    Iron-sulfur clusters: nature's modular, multipurpose structures
    Beinert, Helmut; Holm, Richard H.; Munck, Eckard
    Science (Washington, D. C.) (1997), 277 (5326), 653-659CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    A review with 78 refs. Iron-sulfur proteins are found in all life forms. Most frequently, they contain Fe2S2, Fe3S4, and Fe4S4 clusters. These modular clusters undergo oxidn.-redn. reactions, may be inserted or removed from proteins, can influence protein structure by preferential side chain ligation, and can be interconverted. In addn. to their electron transfer function, iron-sulfur clusters act as catalytic centers and sensors of iron and oxygen. Their most common oxidn. states are paramagnetic and present significant challenges for understanding the magnetic properties of mixed valence systems. Iron-sulfur clusters now rank with such biol. prosthetic groups as hemes and flavins in pervasive occurrence and multiplicity function.
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    Johnson, M. K. Iron–Sulfur Proteins: New Roles for Old Clusters. Curr. Opin. Chem. Biol. 1998, 2, 173– 181,  DOI: 10.1016/S1367-5931(98)80058-6
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    Iron-sulfur proteins: new roles for old clusters
    Johnson, Michael K.
    Current Opinion in Chemical Biology (1998), 2 (2), 173-181CODEN: COCBF4; ISSN:1367-5931. (Current Biology Ltd.)
    A review with 64 refs. Several major advances in our understanding of the structure, function and properties of biol. iron-sulfur clusters have occurred in the past year. These include a new structural type of cluster in the inappropriately named prismane protein, the establishment of redox-mediated [Fe2S2]2+ ↔ [Fe4S4]2+ cluster conversions, and the characterization of valence-delocalized [Fe2S2]+ and all ferrous clusters with [Fe2S2]0, [Fe3S4]2- and [Fe4S4]0 cores. The emergence of novel types of redox, regulatory and enzymic roles have also raised the possibility of iron-sulfur clusters mediating two electron redox processes, coupling proton and electron transfer, and catalyzing disulfide redn. and reductive cleavage of S-adenosylmethionine via sulfur-based cluster chem.
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    Honarmand Ebrahimi, K. Iron-sulfur clusters as inhibitors and catalysts of viral replication. Nat. Chem. 2022, 14 (3), 253– 266,  DOI: 10.1038/s41557-021-00882-0
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    Iron-sulfur clusters as inhibitors and catalysts of viral replication
    Honarmand Ebrahimi, Kourosh; Ciofi-Baffoni, Simone; Hagedoorn, Peter-Leon; Nicolet, Yvain; Le Brun, Nick E.; Hagen, Wilfred R.; Armstrong, Fraser A.
    Nature Chemistry (2022), 14 (3), 253-266CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)
    A review. A virus hijacks host cellular machineries and metabolites in order to reproduce. In response, the innate immune system activates different processes to fight back. Although many aspects of these processes have been well investigated, the key roles played by iron-sulfur [FeS] clusters, which are among the oldest classes of bio-inorg. cofactors, have barely been considered. Here we discuss how several [FeS] cluster-contg. proteins activate, support and modulate the innate immune response to restrict viral infections, and how some of these proteins simultaneously support the replication of viruses. We also propose models of function of some proteins in the innate immune response and argue that [FeS] clusters in many of these proteins act as biol. 'fuses' to control the response. We hope this overview helps to inspire future research in the emerging field of bio-inorg. virol./immunol. and that such studies may reveal new mol. insight into the links between viral infections and diseases like cancer and neurodegeneration.
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    Pritts, J. D.; Michel, S. L. J. Fe-S clusters masquerading as zinc finger proteins. J. Inorg. Biochem 2022, 230, 111756,  DOI: 10.1016/j.jinorgbio.2022.111756
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    Fe-S clusters masquerading as zinc finger proteins
    Pritts, Jordan D.; Michel, Sarah L. J.
    Journal of Inorganic Biochemistry (2022), 230 (), 111756CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Inc.)
    A review. Metal ions are commonly found as protein co-factors in biol., and it is estd. that over a quarter of all proteins require a metal cofactor. The distribution and utilization of metals in biol. has changed over time. As the earth evolved, the atm. became increasingly oxygen rich which affected the bioavailability of certain metals such as iron, which in the oxidized ferric form is significantly less sol. than its reduced ferrous counterpart. Addnl., proteins that utilize metal cofactors for structural purposes grew in abundance, necessitating the use of metal co-factors that are not redox active, such as zinc. One common class of Zn co-factored proteins are zinc finger proteins (ZFs). ZFs bind zinc utilizing cysteine and histidine ligands to promote structure and function. Bioinformatics has annotated 5% of the human genome as ZFs; however, many of these proteins have not been studied empirically. In recent years, examples of annotated ZFs that instead harbor Fe-S clusters have been reported. In this review we highlight four examples of mis-annotated ZFs: mitoNEET, CPSF30, nsp12, and Fep1 and describe methods that can be utilized to differentiate the metal-cofactor.
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    Cameron, J. M.; Janer, A.; Levandovskiy, V.; MacKay, N.; Rouault, T. A.; Tong, W.-H.; Ogilvie, I.; Shoubridge, E. A.; Robinson, B. H. Mutations in iron-sulfur cluster scaffold genes NFU1 and BOLA3 cause a fatal deficiency of multiple respiratory chain and 2-oxoacid dehydrogenase enzymes. Am. J. Hum. Genet. 2011, 89, 486– 495,  DOI: 10.1016/j.ajhg.2011.08.011
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    Mutations in Iron-Sulfur Cluster Scaffold Genes NFU1 and BOLA3 Cause a Fatal Deficiency of Multiple Respiratory Chain and 2-Oxoacid Dehydrogenase Enzymes
    Cameron, Jessie M.; Janer, Alexandre; Levandovskiy, Valeriy; MacKay, Nevena; Rouault, Tracey A.; Tong, Wing-Hang; Ogilvie, Isla; Shoubridge, Eric A.; Robinson, Brian H.
    American Journal of Human Genetics (2011), 89 (4), 486-495CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
    Severe combined deficiency of the 2-oxoacid dehydrogenases, assocd. with a defect in lipoate synthesis and accompanied by defects in complexes I, II, and III of the mitochondrial respiratory chain, is a rare autosomal recessive syndrome with no obvious causative gene defect. A candidate locus for this syndrome was mapped to chromosomal region 2p14 by microcell-mediated chromosome transfer in two unrelated families. Unexpectedly, anal. of genes in this area identified mutations in two different genes, both of which are involved in [Fe-S] cluster biogenesis. A homozygous missense mutation, c.545G>A, near the splice donor of exon 6 in NFU1 predicting a p.Arg182Gln substitution was found in one of the families. The mutation results in abnormal mRNA splicing of exon 6, and no mature protein could be detected in fibroblast mitochondria. A single base-pair duplication c.123dupA was identified in BOLA3 in the second family, causing a frame shift that produces a premature stop codon (p.Glu42Argfs*13). Transduction of fibroblast lines with retroviral vectors expressing the mitochondrial, but not the cytosolic isoform of NFU1 and with isoform 1, but not isoform 2 of BOLA3 restored both respiratory chain function and oxoacid dehydrogenase complexes. NFU1 was previously proposed to be an alternative scaffold to ISCU for the biogenesis of [Fe-S] centers in mitochondria, and the function of BOLA3 was previously unknown. Our results demonstrate that both play essential roles in the prodn. of [Fe-S] centers for the normal maturation of lipoate-contg. 2-oxoacid dehydrogenases, and for the assembly of the respiratory chain complexes.
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    Lossos, A. Fe/S protein assembly gene IBA57 mutation causes hereditary spastic paraplegia. Neurology 2015, 84 (7), 659– 67,  DOI: 10.1212/WNL.0000000000001270
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    Fe/S protein assembly gene IBA57 mutation causes hereditary spastic paraplegia
    Lossos, Alexander; Stuempfig, Claudia; Stevanin, Giovanni; Gaussen, Marion; Zimmerman, Bat-El; Mundwiller, Emeline; Asulin, Moriya; Chamma, Liat; Sheffer, Ruth; Misk, Adel; Dotan, Shlomo; Gomori, John M.; Ponger, Penina; Brice, Alexis; Lerer, Israela; Meiner, Vardiella; Lill, Roland
    Neurology (2015), 84 (7), 659-667CODEN: NEURAI; ISSN:0028-3878. (Lippincott Williams & Wilkins)
    Objective: To present the clin., mol., and cell biol. findings in a family with an autosomal recessive form of hereditary spastic paraplegia characterized by a combination of spastic paraplegia, optic atrophy, and peripheral neuropathy (SPOAN). Methods: We used a combination of whole-genome linkage anal. and exome sequencing to map the disease locus and to identify the responsible gene. To analyze the physiol. consequences of the disease, we used biochem. and cell biol. methods. Results: Ten members of a highly consanguineous family manifested a childhood-onset SPOAN-like phenotype with slow progression into late adulthood. We mapped this disorder to a locus on chromosome 1q and identified a homozygous donor splice-site mutation in the IBA57 gene, previously implicated in 2 infants with lethal perinatal encephalomyopathy. This gene encodes the mitochondrial iron-sulfur (Fe/S) protein assembly factor IBA57. In addn. to a severely decreased amt. of normal IBA57 mRNA, a patient's cells expressed an aberrantly spliced mRNA with a premature stop codon. Lymphoblasts contained 10-fold-lower levels of wild-type, but no signs of truncated IBA57 protein. The decrease in functional IBA57 resulted in reduced levels and activities of several mitochondrial [4Fe-4S] proteins, including complexes I and II, while mitochondrial [2Fe-2S] proteins remained normal. Conclusions: Our findings reinforce the suggested specific function of IBA57 in mitochondrial [4Fe-4S] protein maturation and provide addnl. evidence for its role in human disease. The less decreased IBA57 protein level in this family explains phenotypic differences compared with the previously described lethal encephalomyopathy with no functional IBA57.
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    Debray, F. G. Mutation of the iron-sulfur cluster assembly gene IBA57 causes fatal infantile leukodystrophy. J. Inherit Metab Dis 2015, 38 (6), 1147– 53,  DOI: 10.1007/s10545-015-9857-1
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    Mutation of the iron-sulfur cluster assembly gene IBA57 causes fatal infantile leukodystrophy
    Debray, Francois-Guillaume; Stumpfig, Claudia; Vanlander, Arnaud V.; Dideberg, Vinciane; Josse, Claire; Caberg, Jean-Hubert; Boemer, Francois; Bours, Vincent; Stevens, Rene; Seneca, Sara; Smet, Joel; Lill, Roland; van Coster, Rudy
    Journal of Inherited Metabolic Disease (2015), 38 (6), 1147-1153CODEN: JIMDDP; ISSN:0141-8955. (Springer)
    Leukodystrophies are a heterogeneous group of severe genetic neurodegenerative disorders. A multiple mitochondrial dysfunctions syndrome was found in an infant presenting with a progressive leukoencephalopathy. Homozygosity mapping, whole exome sequencing, and functional studies were used to define the underlying mol. defect. Respiratory chain studies in skeletal muscle isolated from the proband revealed a combined deficiency of complexes I and II. In addn., western blotting indicated lack of protein lipoylation. The combination of these findings was suggestive for a defect in the iron-sulfur (Fe/S) protein assembly pathway. SNP array identified loss of heterozygosity in large chromosomal regions, covering the NFU1 and BOLA3, and the IBA57 and ABCB10 candidate genes, in 2p15-p11.2 and 1q31.1-q42.13, resp. A homozygous c.436C > T (p.Arg146Trp) variant was detected in IBA57 using whole exome sequencing. Complementation studies in a HeLa cell line depleted for IBA57 showed that the mutant protein with the semi-conservative amino acid exchange was unable to restore the biochem. phenotype indicating a loss-of-function mutation of IBA57. In conclusion, defects in the Fe/S protein assembly gene IBA57 can cause autosomal recessive neurodegeneration assocd. with progressive leukodystrophy and fatal outcome at young age. In the affected patient, the biochem. phenotype was characterized by a defect in the respiratory chain complexes I and II and a decrease in mitochondrial protein lipoylation, both resulting from impaired assembly of Fe/S clusters.
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    Iwig, D. F.; Booker, S. J. Insight into the polar reactivity of the onium chalcogen analogues of S-adenosyl-L-methionine. Biochemistry 2004, 43 (42), 13496– 13509,  DOI: 10.1021/bi048693+
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    Insight into the Polar Reactivity of the Onium Chalcogen Analogues of S-Adenosyl-L-methionine
    Iwig, David F.; Booker, Squire J.
    Biochemistry (2004), 43 (42), 13496-13509CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    S-Adenosyl-L-methionine (AdoMet) is one of Nature's most diverse metabolites, used not only in a large no. of biol. reactions but amenable to several different modes of reactivity. The types of transformations in which it is involved include decarboxylation, electrophilic addn. to any of the three carbons bonded to the central sulfur atom, proton removal at carbons adjacent to the sulfonium, and reductive cleavage to generate 5'-deoxyadenosyl 5'-radical intermediates. At physiol. pH and temp., AdoMet is subject to three spontaneous degrdn. pathways, the first of which is racemization of the chiral sulfonium group, which takes place in a pH-independent manner. The two remaining pathways are pH-dependent and include (1) intramol. attack of the α-carboxylate group onto the γ-carbon, affording L-homoserine lactone (HSL) and 5'-methylthioadenosine (MTA), and (2) deprotonation at C-5', initiating a cascade that results in formation of adenine and S-ribosylmethionine. Herein, we describe pH-dependent stability studies of AdoMet and its selenium and tellurium analogs, Se-adenosyl-L-selenomethionine and Te-adenosyl-L-telluromethionine (SeAdoMet and TeAdoMet, resp.), at 37° and const. ionic strength, which we use as a probe of their relative intrinsic reactivities. We find that with AdoMet intramol. nucleophilic attack to afford HSL and MTA exhibits a pH-rate profile having two titratable groups with apparent pKa values of 1.2±0.4 and 8.2±0.05 and displaying first-order rate consts. of <0.7×10-6 s-1 at pH values less than 0.5, ∼3×10-6 s-1 at pH values between 2 and 7, and ∼15×10-6 s-1 at pH values greater than 9. Degrdn. via deprotonation at C-5' follows a pH-rate profile having one titratable group with an apparent pKa value of ∼11.5. The selenium analog decays significantly faster via intramol. nucleophilic attack, also exhibiting a pH-rate profile with two titratable groups with pKa values of ∼0.86 and 8.0±0.1 with first-order rate consts. of <7×10-6 s-1 at pH values less than 0.9, ∼32×10-6 s-1 at pH values between 2 and 7, and ∼170×10-6 s-1 at pH values greater than 9. Degrdn. via deprotonation at C-5' proceeds with one titratable group displaying an apparent pKa value of ∼14.1. Unexpectedly, TeAdoMet did not decay at an observable rate via either of these two pathways. Last, enzymically synthesized AdoMet was found to racemize at rates that were consistent with earlier studies (Hoffman, J. L. (1986) Biochem. 25, 4444-4449); however, SeAdoMet and TeAdoMet did not racemize at detectable rates. In the accompanying paper, we use the information obtained in these model studies to probe the mechanism of cyclopropane fatty acid synthase via use of the onium chalcogens of AdoMet as Me donors.
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    RlmN and AtsB as models for the overproduction and characterization of radical SAM proteins
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    Methods in Enzymology (2012), 516 (Natural Product Biosynthesis by Microorganisms and Plants, Part B), 125-152CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)
    A review. An explosion of remarkable chem. transformations has been witnessed in the past decade as a result of the radical S-adenosyl-L-methionine (SAM) (RS) superfamily of proteins. These proteins share the ability to cleave SAM reductively to L-methionine and a 5'-deoxyadenosyl 5'-radical (5'-dA*). The 5'-dA* initiates >40 distinct reaction types by abstracting target hydrogen atoms on small-mol. and macromol. substrates. All RS enzymes contain a [4Fe-4S] cluster coordinated by SAM that supplies the electron for SAM cleavage. A subset of RS enzymes contains addnl. iron-sulfur (Fe/S) clusters that serve alternative purposes, many remaining to be defined. The oxygen lability of their [4Fe-4S] clusters causes RS enzymes to be more tedious to purify, characterize, and study. Moreover, the type(s) and stoichiometry of Fe/S clusters in RS enzymes has often been a source of debate. Herein, we use RlmN and AtsB as models to highlight methods for purifying and characterizing RS enzymes, focusing on using Mossbauer spectroscopy in concert with methods for quantifying iron and acid-labile sulfide to assign cluster content accurately.
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    Evidence for the formation of a linear [iron-sulfur] ([3Fe-4S]) cluster in partially unfolded aconitase
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    Journal of Biological Chemistry (1984), 259 (23), 14463-71CODEN: JBCHA3; ISSN:0021-9258.
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    Blaszczyk, A. J. Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase. J. Am. Chem. Soc. 2016, 138 (10), 3416– 3426,  DOI: 10.1021/jacs.5b12592
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    Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase
    Blaszczyk, Anthony J.; Silakov, Alexey; Zhang, Bo; Maiocco, Stephanie J.; Lanz, Nicholas D.; Kelly, Wendy L.; Elliott, Sean J.; Krebs, Carsten; Booker, Squire J.
    Journal of the American Chemical Society (2016), 138 (10), 3416-3426CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    TsrM, an annotated radical S-adenosylmethionine (SAM) enzyme, catalyzes the methylation of carbon 2 of the indole ring of L-tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended Me group derives from SAM; however, the enzyme also requires cobalamin and iron-sulfur cluster cofactors for turnover. In this work we report the overprodn. and purifn. of TsrM and the characterization of its metallocofactors by UV-visible, ESR, hyperfine sublevel correlation (HYSCORE), and M.ovrddot.ossbauer spectroscopies as well as protein-film electrochem. (PFE). The enzyme contains 1 equiv of its cobalamin cofactor in its as-isolated state and can be reconstituted with iron and sulfide to contain one [4Fe-4S] cluster with a site-differentiated Fe2+/Fe3+ pair. Our spectroscopic studies suggest that TsrM binds cobalamin in an uncharacteristic five-coordinate base-off/His-off conformation, whereby the dimethylbenzimidazole group is replaced by a non-nitrogenous ligand, which is likely a water mol. Electrochem. anal. of the protein by PFE indicates a one-electron redox feature with a midpoint potential of -550 mV, which is assigned to a [4Fe-4S]2+/[4Fe-4S]+ redox couple. Anal. of TsrM by M.ovrddot.ossbauer and HYSCORE spectroscopies suggests that SAM does not bind to the unique iron site of the cluster in the same manner as in other radical SAM (RS) enzymes, yet its binding still perturbs the electronic configuration of both the Fe/S cluster and the cob(II)alamin cofactors. These biophys. studies suggest that TsrM is an atypical RS enzyme, consistent with its reported inability to catalyze formation of a 5'-deoxyadenosyl 5'-radical.
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This article is cited by 2 publications.

  1. Squire J. Booker, Cody T. Lloyd. Twenty Years of Radical SAM! The Genesis of the Superfamily. ACS Bio & Med Chem Au 2022, 2 (6) , 538-547. https://doi.org/10.1021/acsbiomedchemau.2c00078
  2. Luca Pedroletti, Anna Moseler, Andreas J Meyer, . Assembly, transfer, and fate of mitochondrial iron–sulfur clusters. Journal of Experimental Botany 2023, 25 https://doi.org/10.1093/jxb/erad062
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  • Abstract

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    Figure 1

    Figure 1. Proposed de novo biosynthetic scheme of lipoyl cofactor in humans.

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    Figure 2

    Figure 2. Sequence alignment of LS proteins from E. coli (P60716), M. tuberculosis (P9WK91), T. elongatus (Q8DLC2), and humans (O43766). The conserved residues that ligate the auxiliary and the RS clusters of LS are highlighted in blue and red, respectively.

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    Figure 3

    Figure 3. Biophysical characterization of LIAS: SDS–PAGE gel analysis of the expression and purification of LIAS on a Ni–NTA column (A), UV–vis scan of LIAS (B), Mössbauer spectrum of LIAS (C), and EPR spectrum of dithionite-reduced LIAS (D). (A) Lane 1, pre-IPTG induction; lane 2, post-IPTG induction; lane 3, crude lysate; lane 4, Ni–NTA column flow-through; lane 5, wash; lane 6; SUMO–LIAS fusion eluate; lane 7, LIAS after the SUMO tag is removed; lane 8, protein molecular weight ladder. (B) The UV–vis absorption scan of 8 μM purified LIAS showing a broad absorption at ∼410 nm, which is typical for proteins that bind [4Fe–4S] clusters. (C) The Mössbauer spectrum of 380 μM LIAS at 4.2 K, collected in the presence of a 53 mT external magnetic field applied parallel to the direction of propagation of the γ beam. The vertical bars represent the experimental spectrum, and the blue line shows the features associated with a [4Fe–4S]2+ cluster. The arrow indicates the shoulder resulting from spectral features of a site-differentiated [4Fe–4S]2+ cluster. (D) The EPR spectrum of 400 μM LIAS reduced with 4 mM dithionite and collected at 10 K with 10 mW microwave power and 0.2 mT modulation amplitude confirming bound [4Fe–4S] clusters.

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    Figure 4

    Figure 4. Amino acid sequence alignment of E. coli NfuA (P63020) and human NFU1 (Q9UMS0) showing the two conserved cysteine residues that ligate the Fe–S cluster highlighted in red.

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    Figure 5

    Figure 5. SDS–PAGE analysis of the expression and purification of NFU1 on a Ni–NTA column (A), UV–vis scan of 15 μM NFU1 (B), and Mössbauer spectra of NFU1 (C). (A) Lane 1, pre-IPTG induction; lane 2, post-IPTG induction; lane 3, crude lysate; lane 4, Ni–NTA column flow-through; lane 5, wash; lane 6, SUMO–NFU1 fusion eluate; lane 7, NFU1 after the SUMO tag is removed; lane 8, protein molecular weight ladder. (B) The UV–vis absorption scan spectrum of 15 μM purified NFU1 showing a broad absorption at ∼410 nm indicative of a bound [4Fe–4S] cluster. (C) The 4.2 K Mössbauer spectra of 860 μM NFU1 in the presence of a 53 mT external magnetic field applied parallel to the direction of propagation of the γ beam. The vertical bars represent the experimental spectra, and the blue line shows the features associated with a [4Fe–4S]2+ cluster.

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    Figure 6

    Figure 6. Size-exclusion gel filtration chromatography elution profiles of holo-LIAS (100 μM, blue), holo-NFU1 (200 μM, black), and a 1:1 mixture of holo-LIAS and holo-NFU1 (100 μM each, red) (A). SDS–PAGE analysis of the chromatographed proteins: lane 1, holo-NFU1 alone; lane 2, holo-LIAS alone; lane 3, a mixture of holo-NFU1 and holo-LIAS, indicating complex formation (B). ITC binding results of LIAS titrated into NFU1, showing entropically driven binding with a dissociation constant (KD) of 0.7 ± 0.2 μM (C).

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    Figure 7

    Figure 7. LIAS activity determinations: LIAS (10 μM) activity in the absence of NFU1 (A), in the presence of 200 μM NFU1 (B), in the presence of both 200 μM NFU1 and 5 mM sodium citrate (C), and in the presence of 200 μM NFU1 reconstituted with 34S-labeled sulfide (D). LIAS alone catalyzes about 1 turnover of lipoyl product (blue trace) with the 6-thiooctanoyl intermediate quickly reaching a steady level (black trace) (A). The inclusion of an excess of NFU1 in the LIAS reaction promotes multiple turnovers and generation of more than 5 equiv of lipoyl product (red trace), while the formation and decay of the intermediate mimics that of LIAS alone (gray trace) (B). The inclusion of 5 mM sodium citrate, a divalent metal chelator, does not significantly alter the effect of NFU1 (purple trace) compared to reactions in which citrate is omitted (red trace) (C). In the presence of NFU1 reconstituted with 34S2–, the 32S-labeled lipoyl peptide product is formed first before formation of the mixed 32S–34S (blue trace) and 34S–34S-labeled (red trace) lipoyl peptide products (D). The data in panels C and D suggest direct cluster transfer from NFU1 to LIAS during turnover. Unless otherwise noted, all activity assays included the following at their indicated final concentrations: 350 μM octanoyl peptide substrate, 0.75 mM SAM, and 10 μM SAH nucleosidase. The reactions were carried out at room temperature in a buffer that contained 50 mM HEPES, pH 7.5, and 0.25 M KCl and were initiated with a final concentration of 1 mM dithionite. The respective data shown in panels A–D are averages from assays done in triplicate, and the error bars represent one standard deviation from the mean. The 6-thiooctanoyl intermediate data were fit to an exponential equation that accounts for its formation and decay phases (A and B), while the lipoyl peptide product data were fit to a biphasic double-exponential rate of formation equation, assuming an A → B → C model, as has been previously reported for M. tuberculosis LipA (ref (72)).

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  • References

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      Schonauer, Melissa S.; Kastaniotis, Alexander J.; Kursu, V. A. Samuli; Hiltunen, J. Kalervo; Dieckmann, Carol L.
      Journal of Biological Chemistry (2009), 284 (35), 23234-23242CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Lipoic acid is a sulfur-contg. cofactor required for the function of several multienzyme complexes involved in the oxidative decarboxylation of α-keto acids and glycine. Mechanistic details of lipoic acid metab. are unclear in eukaryotes, despite two well defined pathways for synthesis and covalent attachment of lipoic acid in prokaryotes. Here, the involvement of four genes in the synthesis and attachment of lipoic acid in Saccharomyces cerevisiae is reported. LIP2 and LIP5 are required for lipoylation of all three mitochondrial target proteins: Lat1 and Kgd2, the resp. E2 subunits of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, and Gcv3, the H protein of the glycine cleavage enzyme. LIP3, which encodes a lipoate-protein ligase homolog, is necessary for lipoylation of Lat1 and Kgd2, and the enzymic activity of Lip3 is essential for this function. Finally, GCV3, encoding the H protein target of lipoylation, is itself absolutely required for lipoylation of Lat1 and Kgd2. It is shown that lipoylated Gcv3, and not glycine cleavage activity per se, is responsible for this function. Demonstration that a target of lipoylation is required for lipoylation is a novel result. Through anal. of the role of these genes in protein lipoylation, it is concluded that only one pathway for de novo synthesis and attachment of lipoic acid exists in yeast. A model for protein lipoylation is proposed in which Lip2, Lip3, Lip5, and Gcv3 function in a complex, which may be regulated by the availability of acetyl-CoA, and which in turn may regulate mitochondrial gene expression.
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    7. 7
      Spalding, M. D.; Prigge, S. T. Lipoic acid metabolism in microbial pathogens. Microbiology and molecular biology reviews: MMBR 2010, 74 (2), 200– 228,  DOI: 10.1128/MMBR.00008-10
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      7
      Lipoic acid metabolism in microbial pathogens
      Spalding, Maroya D.; Prigge, Sean T.
      Microbiology and Molecular Biology Reviews (2010), 74 (2), 200-228CODEN: MMBRF7; ISSN:1092-2172. (American Society for Microbiology)
      A review. Lipoic acid [(R)-5-(1,2-dithiolan-3-yl)pentanoic acid] is an enzyme cofactor required for intermediate metab. in free-living cells. Lipoic acid was discovered nearly 60 years ago and was shown to be covalently attached to proteins in several multicomponent dehydrogenases. Cells can acquire lipoate (the deprotonated charge form of lipoic acid that dominates at physiol. pH) through either scavenging or de novo synthesis. Microbial pathogens implement these basic lipoylation strategies with a surprising variety of adaptations which can affect pathogenesis and virulence. Similarly, lipoylated proteins are responsible for effects beyond their classical roles in catalysis. These include roles in oxidative defense, bacterial sporulation, and gene expression. This review surveys the role of lipoate metab. in bacterial, fungal, and protozoan pathogens and how these organisms have employed this metab. to adapt to niche environments.
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    8. 8
      Cronan, J. E. Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation. EcoSal Plus 2014, 6 (1), 0001-2012,  DOI: 10.1128/ecosalplus.ESP-0001-2012
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    9. 9
      Ewald, R. Lipoate-Protein Ligase and Octanoyltransferase Are Essential for Protein Lipoylation in Mitochondria of Arabidopsis. Plant Physiology 2014, 165 (3), 978– 990,  DOI: 10.1104/pp.114.238311
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      9
      Lipoate-protein ligase and octanoyltransferase are essential for protein lipoylation in mitochondria of Arabidopsis
      Ewald, Ralph; Hoffmann, Christiane; Florian, Alexandra; Neuhaus, Ekkehard; Fernie, Alisdair R.; Bauwe, Hermann
      Plant Physiology (2014), 165 (3), 978-990CODEN: PLPHAY; ISSN:0032-0889. (American Society of Plant Biologists)
      Prosthetic lipoyl groups are required for the function of several essential multienzyme complexes, such as pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGDH), and the glycine cleavage system (glycine decarboxylase [GDC]). How these proteins are lipoylated has been extensively studied in prokaryotes and yeast (Saccharomyces cerevisiae), but little is known for plants. We earlier reported that mitochondrial fatty acid synthesis by ketoacyl-acyl carrier protein synthase is not vital for protein lipoylation in Arabidopsis (Arabidopsis thaliana) and does not play a significant role in roots. Here, we identify Arabidopsis lipoate-protein ligase (AtLPLA) as an essential mitochondrial enzyme that uses octanoyl-nucleoside monophosphate and possibly other donor substrates for the octanoylation of mitochondrial PDH-E2 and GDC H-protein; it shows no reactivity with bacterial and possibly plant KGDH-E2. The octanoate-activating enzyme is unknown, but we assume that it uses octanoyl moieties provided by mitochondrial β-oxidn. AtLPLA is essential for the octanoylation of PDH-E2, whereas GDC H-protein can optionally also be octanoylated by octanoyltransferase (LIP2) using octanoyl chains provided by mitochondrial ketoacyl-acyl carrier protein synthase to meet the high lipoate requirement of leaf mesophyll mitochondria. Similar to protein lipoylation in yeast, LIP2 likely also transfers octanoyl groups attached to the H-protein to KGDH-E2 but not to PDH-E2, which is exclusively octanoylated by LPLA. We suggest that LPLA and LIP2 together provide a basal protein lipoylation network to plants that is similar to that in other eukaryotes.
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    10. 10
      Cronan, J. E. Assembly of lipoic acid on its cognate enzymes: an extraordinary and essential biosynthetic pathway. Microbiol. Mol. Biol. Rev. 2016, 80, 429– 450,  DOI: 10.1128/MMBR.00073-15
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      10
      Assembly of lipoic acid on its cognate enzymes: an extraordinary and essential biosynthetic pathway
      Cronan, John E.
      Microbiology and Molecular Biology Reviews (2016), 80 (2), 429-450CODEN: MMBRF7; ISSN:1098-5557. (American Society for Microbiology)
      Although the structure of lipoic acid and its role in bacterial metab. were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become understood. Unlike most cofactors, lipoic acid must be covalently bound to its cognate enzyme proteins (the 2-oxoacid dehydrogenases and the glycine cleavage system) in order to function in central metab. Indeed, the cofactor is assembled on its cognate proteins rather than being assembled and subsequently attached as in the typical pathway, like that of biotin attachment. The first lipoate biosynthetic pathway detd. was that of Escherichia coli, which utilizes two enzymes to form the active lipoylated protein from a fatty acid biosynthetic intermediate. Recently, a more complex pathway requiring four proteins was discovered in Bacillus subtilis, which is probably an evolutionary relic. This pathway requires the H protein of the glycine cleavage system of single-carbon metab. to form active (lipoyl) 2-oxoacid dehydrogenases. The bacterial pathways inform the lipoate pathways of eukaryotic organisms. Plants use the E. coli pathway, whereas mammals and fungi probably use the B. subtilis pathway. The lipoate metab. enzymes (except those of sulfur insertion) are members of PFAM family PF03099 (the cofactor transferase family). Although these enyzmes share some sequence similarity, they catalyze three markedly distinct enzyme reactions, making the usual assignment of function based on alignments prone to frequent mistaken annotations. This state of affairs has possibly clouded the interpretation of one of the disorders of human lipoate metab.
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    11. 11
      Solmonson, A.; DeBerardinis, R. J. Lipoic acid metabolism and mitochondrial redox regulation. J. Biol. Chem. 2018, 293, 7522– 7530,  DOI: 10.1074/jbc.TM117.000259
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      11
      Lipoic acid metabolism and mitochondrial redox regulation
      Solmonson, Ashley; De Berardinis, Ralph J.
      Journal of Biological Chemistry (2018), 293 (20), 7522-7530CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      A review. Lipoic acid is an essential cofactor for mitochondrial metab. and is synthesized de novo using intermediates from mitochondrial fatty-acid synthesis type II, S-adenosylmethionine and iron-sulfur clusters. This cofactor is required for catalysis by multiple mitochondrial 2-ketoacid dehydrogenase complexes, including pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase. Lipoic acid also plays a crit. role in stabilizing and regulating these multienzyme complexes. Many of these dehydrogenases are regulated by reactive oxygen species, mediated through the disulfide bond of the prosthetic lipoyl moiety. Collectively, its functions explain why lipoic acid is required for cell growth, mitochondrial activity, and coordination of fuel metab.
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    12. 12
      Reed, L. J. A trail of research from lipoic acid to -keto acid dehydrogenase complexes. J. Biol. Chem. 2001, 276, 38329– 38336,  DOI: 10.1074/jbc.R100026200
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      12
      A trail of research from lipoic acid to α-keto acid dehydrogenase complexes
      Reed, Lester J.
      Journal of Biological Chemistry (2001), 276 (42), 38329-38336CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      A review, retracing the trail of research that began with the isolation and characterization of a microbial growth factor and led to elucidation of the structure, function, and regulation of α-keto acid dehydrogenase complexes.
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    13. 13
      Mayr, J. A.; Feichtinger, R. G.; Tort, F.; Ribes, A.; Sperl, W. Lipoic acid biosynthesis defects. J. Inherit. Metab. Dis. 2014, 37 (4), 553– 563,  DOI: 10.1007/s10545-014-9705-8
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      13
      Lipoic acid biosynthesis defects
      Mayr, Johannes A.; Feichtinger, Rene G.; Tort, Frederic; Ribes, Antonia; Sperl, Wolfgang
      Journal of Inherited Metabolic Disease (2014), 37 (4), 553-563CODEN: JIMDDP; ISSN:0141-8955. (Springer)
      A review. Lipoate is a covalently bound cofactor essential for five redox reactions in humans: in four 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). Two enzymes are from the energy metab., α-ketoglutarate dehydrogenase and pyruvate dehydrogenase; and three are from the amino acid metab., branched-chain ketoacid dehydrogenase, 2-oxoadipate dehydrogenase, and the GCS. All these enzymes consist of multiple subunits and share a similar architecture. Lipoate synthesis in mitochondria involves mitochondrial fatty acid synthesis up to octanoyl-acyl-carrier protein; and three lipoate-specific steps, including octanoic acid transfer to glycine cleavage H protein by lipoyl(octanoyl) transferase 2 (putative) (LIPT2), lipoate synthesis by lipoic acid synthetase (LIAS), and lipoate transfer by lipoyltransferase 1 (LIPT1), which is necessary to lipoylate the E2 subunits of the 2-oxoacid dehydrogenases. The reduced form dihydrolipoate is reactivated by dihydrolipoyl dehydrogenase (DLD). Mutations in LIAS have been identified that result in a variant form of nonketotic hyperglycinemia with early-onset convulsions combined with a defect in mitochondrial energy metab. with encephalopathy and cardiomyopathy. LIPT1 deficiency spares the GCS, and resulted in a combined 2-oxoacid dehydrogenase deficiency and early death in one patient and in a less severely affected individual with a Leigh-like phenotype. As LIAS is an iron-sulfur-cluster-dependent enzyme, a no. of recently identified defects in mitochondrial iron-sulfur cluster synthesis, including NFU1, BOLA3, IBA57, GLRX5 presented with deficiency of LIAS and a LIAS-like phenotype. As in DLD deficiency, a broader clin. spectrum can be anticipated for lipoate synthesis defects depending on which of the affected enzymes is most rate limiting.
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    14. 14
      Habarou, F.; Hamel, Y.; Haack, T. B.; Feichtinger, R. G.; Lebigot, E.; Marquardt Biallelic mutations in LIPT2 cause a mitochondrial lipoylation defect associated with severe neonatal encephalopathy. Am. J. Hum. Genet. 2017, 101, 283– 290,  DOI: 10.1016/j.ajhg.2017.07.001
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      14
      Biallelic Mutations in LIPT2 Cause a Mitochondrial Lipoylation Defect Associated with Severe Neonatal Encephalopathy
      Habarou, Florence; Hamel, Yamina; Haack, Tobias B.; Feichtinger, Rene G.; Lebigot, Elise; Marquardt, Iris; Busiah, Kanetee; Laroche, Cecile; Madrange, Marine; Grisel, Coraline; Pontoizeau, Clement; Eisermann, Monika; Boutron, Audrey; Chretien, Dominique; Chadefaux-Vekemans, Bernadette; Barouki, Robert; Bole-Feysot, Christine; Nitschke, Patrick; Goudin, Nicolas; Boddaert, Nathalie; Nemazanyy, Ivan; Delahodde, Agnes; Kolker, Stefan; Rodenburg, Richard J.; Korenke, G. Christoph; Meitinger, Thomas; Strom, Tim M.; Prokisch, Holger; Rotig, Agnes; Ottolenghi, Chris; Mayr, Johannes A.; de Lonlay, Pascale
      American Journal of Human Genetics (2017), 101 (2), 283-290CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
      Lipoate serves as a cofactor for the glycine cleavage system (GCS) and four 2-oxoacid dehydrogenases functioning in energy metab. (α-oxoglutarate dehydrogenase [α-KGDHc] and pyruvate dehydrogenase [PDHc]), or amino acid metab. (branched-chain oxoacid dehydrogenase, 2-oxoadipate dehydrogenase). Mitochondrial lipoate synthesis involves three enzymic steps catalyzed sequentially by lipoyl(octanoyl) transferase 2 (LIPT2), lipoic acid synthetase (LIAS), and lipoyltransferase 1 (LIPT1). Mutations in LIAS have been assocd. with nonketotic hyperglycinemia-like early-onset convulsions and encephalopathy combined with a defect in mitochondrial energy metab. LIPT1 deficiency spares GCS deficiency and has been assocd. with a biochem. signature of combined 2-oxoacid dehydrogenase deficiency leading to early death or Leigh-like encephalopathy. We report on the identification of biallelic LIPT2 mutations in three affected individuals from two families with severe neonatal encephalopathy. Brain MRI showed major cortical atrophy with white matter abnormalities and cysts. Plasma glycine was mildly increased. Affected individuals' fibroblasts showed reduced oxygen consumption rates, PDHc, α-KGDHc activities, leucine catabolic flux, and decreased protein lipoylation. A normalization of lipoylation was obsd. after expression of wild-type LIPT2, arguing for LIPT2 requirement in intramitochondrial lipoate synthesis. Lipoic acid supplementation did not improve clin. condition nor activities of PDHc, α-KGDHc, or leucine metab. in fibroblasts and was ineffective in yeast deleted for the orthologous LIP2.
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    15. 15
      Cao, X.; Zhu, L.; Song, X.; Hu, Z.; Cronan, J. E. Protein moonlighting elucidates the essential human pathway catalyzing lipoic acid assembly on its cognate enzymes. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (30), E7063– E7072,  DOI: 10.1073/pnas.1805862115
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      15
      Protein moonlighting elucidates the essential human pathway catalyzing lipoic acid assembly on its cognate enzymes
      Cao, Xinyun; Zhu, Lei; Song, Xuejiao; Hu, Zhe; Cronan, John E.
      Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (30), E7063-E7072CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The lack of attachment of lipoic acid to its cognate enzyme proteins results in devastating human metabolic disorders. These mitochondrial disorders are evident soon after birth and generally result in early death. The mutations causing specific defects in lipoyl assembly map in three genes, LIAS, LIPT1, and LIPT2. Although physiol. roles have been proposed for the encoded proteins, only the LIPT1 protein had been studied at the enzyme level. LIPT1 was reported to catalyze only the second partial reaction of the classical lipoate ligase mechanism. We report that the physiol. relevant LIPT1 enzyme activity is transfer of lipoyl moieties from the H protein of the glycine cleavage system to the E2 subunits of the 2-oxoacid dehydrogenases required for respiration (e.g., pyruvate dehydrogenase) and amino acid degrdn. We also report that LIPT2 encodes an octanoyl transferase that initiates lipoyl group assembly. The human pathway is now biochem. defined.
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    16. 16
      Parry, R. J. Biosynthesis of lipoic acid. 1. Incorporation of specifically tritiated octanoic acid into lipoic acid. J. Am. Chem. Soc. 1977, 99 (19), 6464– 6466,  DOI: 10.1021/ja00461a061
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      16
      Biosynthesis of lipoic acid. 1. Incorporation of specifically tritiated octanoic acid into lipoic acid
      Parry, Ronald J.
      Journal of the American Chemical Society (1977), 99 (19), 6464-6CODEN: JACSAT; ISSN:0002-7863.
      The mechanism of the conversion of octanoate into lipoate by Escherichia coli was examd. by administration of octanoic acid-carboxyl-14C to E. coli in conjunction with tritiated forms of octanoic acid labeled specifically at those C atoms which might be involved in the introduction of S. No 3H loss occurs from the methylene groups of octanoic acid adjacent to the sites of S introduction. Introduction of S at C-6 of octanoate proceeds with ∼50% 3H loss and introduction of S at C-8 of octanoate proceeds without significant loss of 3H.
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      White, R. H. Stable isotope studies on the biosynthesis of lipoic acid in Escherichia coli. Biochemistry 1980, 19 (1), 15– 19,  DOI: 10.1021/bi00542a003
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      17
      Stable isotope studies on the biosynthesis of lipoic acid in Escherichia coli
      White, Robert H.
      Biochemistry (1980), 19 (1), 15-19CODEN: BICHAW; ISSN:0006-2960.
      A method was developed for the gas chromatog.-mass spectrometric (GC-MS) identification of lipoic acid in tissue. The method consists of acid hydrolysis of the tissue to free the bound lipoic acid, methylene chloride extn. of the lipoic acid, and subsequent chem. derivatization of the lipoic acid as Me 6,8-bis(benzylthio)octanoate prior to GC-MS anal. By use of this method of anal., the incorporation of deuterium into lipoic acid by E. coli growing on acetate-methyl-2H3 was studied. The results showed that the lipoic acid was biosynthesized from octanoic acid with the loss of only 1 deuterium-contg. position at C-8. The deuterium incorporated at C-6 of octanoic acid from the labeled acetate was retained. Since this deuterium is incorporated in the L-configuration during fatty acid biosynthesis and is known to have the D-configuration in lipoic acid, it is concluded that an inversion of configuration occurs at C-6 during the sulfur insertion.
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      Jordan, S. W.; Cronan, J. E., Jr. A new metabolic link. J. Biol. Chem. 1997, 272, 17903– 17906,  DOI: 10.1074/jbc.272.29.17903
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      18
      A new metabolic link. The acyl carrier protein of lipid synthesis donates lipoic acid to the pyruvate dehydrogenase complex in Escherichia coli and mitochondria
      Jordan, Sean W.; Cronan, John E., Jr.
      Journal of Biological Chemistry (1997), 272 (29), 17903-17906CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Lipoic acid is an essential enzyme cofactor that requires covalent attachment to its cognate proteins to confer biol. activity. The major lipoylated proteins are highly conserved enzymes of central metab., the pyruvate and α-ketoglutarate dehydrogenase complexes. The classical lipoate ligase uses ATP to activate the lipoate carboxy group followed by attachment of the cofactor to a specific subunit of each dehydrogenase complex, and it was assumed that all lipoate attachment preceded by this mechanism. However, our previous work indicated in the absence of detectable ATP-dependent ligase activity raising the possibility of a class of enzyme that attaches lipoate to the dehydrogenase complexes by a different mechanism. We now report that E. coli and mitochondria contain lipoate transferases that use lipoyl-acyl carrier as the lipoate donor. This finding demonstrates a direct link between fatty acid synthesis and lipoate attachment an also provides the first direct demonstration of a role for the enigmatic acyl carrier proteins of mitochondria.
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      Jordan, S. W.; Cronan, J. E. [19] Biosynthesis of lipoic acid and posttranslational modification with lipoic acid in Escherichia coli. Methods Enzymol. 1997, 279, 176– 183,  DOI: 10.1016/S0076-6879(97)79021-9
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      19
      Biosynthesis of lipoic acid and posttranslational modification with lipoic acid in Escherichia coli
      Jordan, Sean W.; Cronan, John E., Jr.
      Methods in Enzymology (1997), 279 (Vitamins and Coenzymes, Part I), 176-183CODEN: MENZAU; ISSN:0076-6879. (Academic)
      A report is given on the biosynthetic pathway of lipoic acid and the posttranslational modification of proteins by lipoate ligases in E. coli.
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    20. 20
      Jordan, S. W.; Cronan, J. E. The Escherichia coli lipB Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase. J. Bacteriol. 2003, 185 (5), 1582– 1589,  DOI: 10.1128/JB.185.5.1582-1589.2003
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      20
      The Escherichia coli lipB gene encodes lipoyl (octanoyl)-acyl carrier protein:protein transferase
      Jordan, Sean W.; Cronan, John E., Jr.
      Journal of Bacteriology (2003), 185 (5), 1582-1589CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      In an earlier study we reported a new enzyme, lipoyl-[acyl carrier protein]-protein N-lipoyltransferase, in Escherichia coli and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase. It was also shown that E. coli lipB mutants lack this enzyme activity, a finding consistent with lipB being the gene that encoded the lipoyltransferase. However, it remained possible that lipB encoded a pos. regulator required for lipoyltransferase expression or action. We now report genetic and biochem. evidence demonstrating that lipB encodes the lipoyltransferase. A lipB temp.-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the lipB-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. In the course of these expts. the ATG initiation codon commonly assigned to lipB genes in genomic databases was shown to produce a nonfunctional E. coli LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymically active protein. Prior genetic results suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addn. to its normal substrates lipoic acid plus ATP. We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. A nonhydrolyzable lipoyl-AMP analog was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chem. diverse reactions.
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      Nesbitt, N. M. Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase. Protein Expr Purif 2005, 39 (2), 269– 82,  DOI: 10.1016/j.pep.2004.10.021
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      Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase
      Nesbitt, Natasha M.; Baleanu-Gogonea, Camelia; Cicchillo, Robert M.; Goodson, Kathy; Iwig, David F.; Broadwater, John A.; Haas, Jeffrey A.; Fox, Brian G.; Booker, Squire J.
      Protein Expression and Purification (2005), 39 (2), 269-282CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier)
      Lipoic acid is a sulfur-contg. 8-carbon fatty acid that functions as a central cofactor in multienzyme complexes that are involved in the oxidative decarboxylation of glycine and several α-keto acids. In its functional form, it is bound covalently in an amide linkage to the ε-amino group of a conserved lysine residue of the "lipoyl bearing subunit," resulting in a long "swinging arm" that shuttles intermediates among the requisite active sites. In Escherichia coli and many other organisms, the lipoyl cofactor can be synthesized endogenously. The 8-carbon fatty-acyl chain is constructed via the type II fatty acid biosynthetic pathway as an appendage to the acyl carrier protein (ACP). Lipoyl(octanoyl)transferase (LipB) transfers the octanoyl chain from ACP to the target lysine acceptor, generating the substrate for lipoyl synthase (LS), which subsequently catalyzes insertion of both sulfur atoms into the C-6 and C-8 positions of the octanoyl chain. In this study, we present a three-step isolation procedure that results in a 14-fold purifn. of LipB to >95% homogeneity in an overall yield of 25%. We also show that the protein catalyzes the transfer of the octanoyl group from octanoyl-ACP to apo-H protein, which is the lipoyl bearing subunit of the glycine cleavage system. The specific activity of the purified protein is 0.541 U mg-1, indicating a turnover no. of ∼0.2 s-1, and the apparent Km values for octanoyl-ACP and apo-H protein are 10.2±4.4 and 13.2±2.9 μM, resp.
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      Parry, R. J.; Trainor, D. A. Biosynthesis of lipoic acid. 2. Stereochemistry of sulfur introduction at C-6 of octanoic acid. J. Am. Chem. Soc. 1978, 100 (16), 5243– 5244,  DOI: 10.1021/ja00484a073
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      22
      Biosynthesis of lipoic acid. 2. Stereochemistry of sulfur introduction at C-6 of octanoic acid
      Parry, Ronald J.; Trainor, Diane A.
      Journal of the American Chemical Society (1978), 100 (16), 5243-4CODEN: JACSAT; ISSN:0002-7863.
      (6R)- and (6S)-Octanoic acid-3H were synthesized and their incorporation into lipoic acid by Escherichia coli was investigated. The precursor incorporation expts. demonstrated that the 6-pro-R H atom of octanoic acid is lost as the result of S atom introduction and that the S atom introduction therefore proceeds with inversion of configuration.
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    23. 23
      Reed, K. E.; Cronan, J. E. Lipoic acid metabolism in Escherichia coli: sequencing and functional characterization of the lipA and lipB genes. J. Bacteriol. 1993, 175 (5), 1325– 1336,  DOI: 10.1128/jb.175.5.1325-1336.1993
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      23
      Lipoic acid metabolism in Escherichia coli: Sequencing and functional characterization of the lipA and lipB genes
      Reed, Kelynne E.; Cronan, John E., Jr.
      Journal of Bacteriology (1993), 175 (5), 1325-36CODEN: JOBAAY; ISSN:0021-9193.
      Two genes, lipA and lipB, involved in lipoic acid biosynthesis or metab. were characterized by DNA sequence anal. The translational initiation site of the lipA gene was established, and the lipB gene product was identified as a 15-kDa protein. Overprodn. of LipA resulted in the formation of inclusion bodies, from which the protein was readily purified. Cells grown under strictly anaerobic conditions required the lipA and lipB gene products for the synthesis of a functional glycine cleavage system. Mutants carrying a null mutation in the lipB gene retained a partial ability to synthesize lipoic acid and produced low levels of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase activities. The lipA gene product failed to convert protein-bound octanoic acid moieties to lipoic acid moieties in vivo; however, the growth of both lipA and lipB mutants was supported by either 6-thiooctanoic acid or 8-thiooctanoic acid in place of lipoic acid. These data suggest that LipA is required from the insertion of the first sulfur into the octanoic acid backbone. LipB functions downstream of LipA, but its role in lipoic acid metab. remains unclear.
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      Miller, J. R. Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein. Biochemistry 2000, 39, 15166– 15178,  DOI: 10.1021/bi002060n
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      Escherichia coli LipA Is a Lipoyl Synthase: In Vitro Biosynthesis of Lipoylated Pyruvate Dehydrogenase Complex from Octanoyl-Acyl Carrier Protein
      Miller, J. Richard; Busby, Robert W.; Jordan, Sean W.; Cheek, Jennifer; Henshaw, Timothy F.; Ashley, Gary W.; Broderick, Joan B.; Cronan, John E., Jr.; Marletta, Michael A.
      Biochemistry (2000), 39 (49), 15166-15178CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been obsd. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as sol. proteins. The proteins were purified as a mixt. of monomeric and dimeric species that contain approx. four iron atoms per LipA polypeptide and a similar amt. of acid-labile sulfide. ESR and electronic absorbance spectroscopy indicate that the proteins contain a mixt. of [3Fe-4S] and [4Fe-4S] cluster states. Redn. with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S]1+ cluster with the majority of the protein contg. a species consistent with an S = 0 [4Fe-4S]2+ cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), resp., to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chem.
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      Cicchillo, R. M. Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid. Biochemistry 2004, 43 (21), 6378– 86,  DOI: 10.1021/bi049528x
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      Lipoyl Synthase Requires Two Equivalents of S-Adenosyl-L-methionine To Synthesize One Equivalent of Lipoic Acid
      Cicchillo, Robert M.; Iwig, David F.; Jones, A. Daniel; Nesbitt, Natasha M.; Baleanu-Gogonea, Camelia; Souder, Matthew G.; Tu, Loretta; Booker, Squire J.
      Biochemistry (2004), 43 (21), 6378-6386CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various α-keto acids, as well as the cleavage of glycine into CO2 and NH3, with concomitant transfer of its α-carbon to tetrahydrofolate, generating N5,N10-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochem. studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-L-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA•), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming L-methionine in addn. to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated deriv. of the lipoyl-bearing subunit (E2) of the pyruvate dehydrogenase complex. Herein, the authors show that the octanoylated deriv. of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, the authors show that the 5'-dA• acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d15]H-protein to 5'-deoxyadenosine. Last, the authors' data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.
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      Oberg, N. RadicalSAM.org: A Resource to Interpret Sequence-Function Space and Discover New Radical SAM Enzyme Chemistry. ACS Bio & Med. Chem. Au 2022, 2 (1), 22– 35,  DOI: 10.1021/acsbiomedchemau.1c00048
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      RadicalSAM.org: A Resource to Interpret Sequence-Function Space and Discover New Radical SAM Enzyme Chemistry
      Oberg, Nils; Precord, Timothy W.; Mitchell, Douglas A.; Gerlt, John A.
      ACS Bio & Med Chem Au (2022), 2 (1), 22-35CODEN: ABMCB8; ISSN:2694-2437. (American Chemical Society)
      The radical SAM superfamily (RSS), arguably the most functionally diverse enzyme superfamily, is also one of the largest with ~ 700 K members currently in the UniProt database. The vast majority of the members have uncharacterized enzymic activities and metabolic functions. In this Perspective, we describe RadicalSAM.org, a new web-based resource that enables a user-friendly genomic enzymol. strategy to explore sequence-function space in the RSS. The resource attempts to enable identification of isofunctional groups of radical SAM enzymes using sequence similarity networks (SSNs) and the genome context of the bacterial, archaeal, and fungal members provided by genome neighborhood diagrams (GNDs). Enzymic activities and in vivo functions frequently can be inferred from genome context given the tendency for genes of related function to be clustered. We invite the scientific community to use RadicalSAM.org to (i) guide their exptl. studies to discover new enzymic activities and metabolic functions, (ii) contribute exptl. verified annotations to RadicalSAM.org to enhance the ability to predict novel activities and functions, and (iii) provide suggestions for improving this resource.
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      Broderick, J. B. Radical S-Adenosylmethionine Enzymes. Chem. Rev. 2014, 114 (8), 4229– 4317,  DOI: 10.1021/cr4004709
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      Radical S-adenosylmethionine enzymes
      Broderick, Joan B.; Duffus, Benjamin R.; Duschene, Kaitlin S.; Shepard, Eric M.
      Chemical Reviews (Washington, DC, United States) (2014), 114 (8), 4229-4317CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The review begins by summarizing unifying features of radical S-adenosylmethionine (SAM) enzymes, and subsequent sections delve into the biochem., spectroscopic, structural, and mechanistic details for those enzymes that have been characterized.
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      Landgraf, B. J.; McCarthy, E. L.; Booker, S. J. Radical S-Adenosylmethionine Enzymes in Human Health and Disease. Annu. Rev. Biochem. 2016, 85 (1), 485– 514,  DOI: 10.1146/annurev-biochem-060713-035504
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      Radical S-Adenosylmethionine Enzymes in Human Health and Disease
      Landgraf, Bradley J.; McCarthy, Erin L.; Booker, Squire J.
      Annual Review of Biochemistry (2016), 85 (), 485-514CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
      A review. Radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chem. challenging reactions across all domains of life. Of approx. 114,000 of these enzymes, 8 are known to be present in humans: MOCS1, molybdenum cofactor biosynthesis; LIAS, lipoic acid biosynthesis; CDK5RAP1, 2-methylthio-N6-isopentenyladenosine biosynthesis; CDKAL1, methylthio-N6-threonylcarbamoyladenosine biosynthesis; TYW1, wybutosine biosynthesis; ELP3, 5-methoxycarbonylmethyl uridine; and RSAD1 and viperin, both of unknown function. Aberrations in the genes encoding these proteins result in a variety of diseases. In this review, we summarize the biochem. characterization of these 8 radical S-adenosylmethionine enzymes and, in the context of human health, describe the deleterious effects that result from such genetic mutations.
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      Holliday, G. L. Atlas of the Radical SAM Superfamily: Divergent Evolution of Function Using a ″Plug and Play″ Domain. Methods Enzymol 2018, 606, 1– 71,  DOI: 10.1016/bs.mie.2018.06.004
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      Atlas of the radical SAM superfamily: divergent evolution of function using a "plug and play" domain
      Holliday, Gemma L.; Akiva, Eyal; Meng, Elaine C.; Brown, Shoshana D.; Calhoun, Sara; Pieper, Ursula; Sali, Andrej; Booker, Squire J.; Babbitt, Patricia C.
      Methods in Enzymology (2018), 606 (Radical SAM Enzymes), 1-71CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)
      The radical SAM superfamily contains over 100,000 homologous enzymes that catalyze a remarkably broad range of reactions required for life, including metab., nucleic acid modification, and biogenesis of cofactors. While the highly conserved SAM-binding motif responsible for formation of the key 5'-deoxyadenosyl radical intermediate is a key structural feature that simplifies identification of superfamily members, our understanding of their structure-function relationships is complicated by the modular nature of their structures, which exhibit varied and complex domain architectures. To gain new insight about these relationships, we classified the entire set of sequences into similarity-based subgroups that could be visualized using sequence similarity networks. This superfamily-wide anal. reveals important features that had not previously been appreciated from studies focused on one or a few members. Functional information mapped to the networks indicates which members have been exptl. or structurally characterized, their known reaction types, and their phylogenetic distribution. Despite the biol. importance of radical SAM chem., the vast majority of superfamily members have never been exptl. characterized in any way, suggesting that many new reactions remain to be discovered. In addn. to 20 subgroups with at least one known function, we identified addnl. subgroups made up entirely of sequences of unknown function. Importantly, our results indicate that even general reaction types fail to track well with our sequence similarity-based subgroupings, raising major challenges for function prediction for currently identified and new members that continue to be discovered. Interactive similarity networks and other data from this anal. are available from the Structure-Function Linkage Database.
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      Booker, S. J.; Grove, T. L. Mechanistic and functional versatility of radical SAM enzymes. F1000 Biol. Rep. 2010, 2, 52,  DOI: 10.3410/B2-52
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      Mechanistic and functional versatility of radical SAM enzymes
      Booker Squire J; Grove Tyler L
      F1000 biology reports (2010), 2 (), 52 ISSN:.
      Enzymes of the radical SAM (RS) superfamily catalyze a diverse assortment of reactions that proceed via intermediates containing unpaired electrons. The radical initiator is the common metabolite S-adenosyl-l-methionine (SAM), which is reductively cleaved to generate a 5'-deoxyadenosyl 5'-radical, a universal and obligate intermediate among enzymes within this class. A bioinformatics study that appeared in 2001 indicated that this superfamily contained over 600 members, many catalyzing reactions that were rich in novel chemical transformations. Since that seminal study, the RS superfamily has grown immensely, and new details about the scope of reactions and biochemical pathways in which its members participate have emerged. This review will highlight only a few of the most significant findings from the past 2-3 years, focusing primarily on: RS enzymes involved in complex metallocofactor maturation; characterized RS enzymes that lack the canonical CxxxCxxC motif; RS enzymes containing multiple iron-sulfur clusters; RS enzymes catalyzing reactions with compelling medical implications; and the energetics and mechanism of generating the 5'-deoxyadenosyl radical. A number of significant studies of RS enzymes will unfortunately be omitted, and it is hoped that the reader will access the relevant literature - particularly a number of superb review articles recently written on the subject - to acquire a deeper appreciation of this class of enzymes.
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      Bandarian, V. Journey on the Radical SAM Road as an Accidental Pilgrim. ACS Bio Med Chem Au 2022,  DOI: 10.1021/acsbiomedchemau.1c00059
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      Frey, P. A.; Booker, S. J. Radical mechanisms of S-adenosylmethionine-dependent enzymes. Adv. Protein Chem. 2001, 58, 1– 45,  DOI: 10.1016/S0065-3233(01)58001-8
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      Radical mechanisms of S-adenosylmethionine-dependent enzymes
      Frey, Perry A.; Booker, Squire J.
      Advances in Protein Chemistry (2001), 58 (Novel Cofactors), 1-45CODEN: APCHA2; ISSN:0065-3233. (Academic Press)
      A review. The authors describe the family of S-adenosylmethionine (SAM)-dependent enzymes that make use of the 5'-deoxyadenosyl radical, with special ref. to the mechanism by which SAM is cleaved reversibly at the active site. They also consider the functions of the 5'-deoxyadenosyl radical and the mechanisms of these diverse reactions. Special consideration is given to lysine 2,3-aminomutase, pyruvate formate lyase, anaerobic ribonucleotide reductase and biotin synthase. (c) 2001 Academic Press.
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      Frey, P. A.; Hegeman, A. D.; Ruzicka, F. J. The Radical SAM Superfamily. Crit Rev. Biochem Mol. Biol. 2008, 43 (1), 63– 88,  DOI: 10.1080/10409230701829169
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      The Radical SAM Superfamily
      Frey, Perry A.; Hegeman, Adrian D.; Ruzicka, Frank J.
      Critical Reviews in Biochemistry and Molecular Biology (2008), 43 (1), 63-88CODEN: CRBBEJ; ISSN:1040-9238. (Informa Healthcare)
      A review. The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The characteristic three-cysteine motif nucleates a [4Fe-4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochem. transformations, and most members have not been biochem. characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochem. information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochem. characterized to date have in common the cleavage of the [4Fe-4S]1 + -SAM complex to [4Fe-4S]2 +-Met and the 5' -deoxyadenosyl radical, which abstrs. a hydrogen atom from the substrate to initiate a radical mechanism.
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      Cicchillo, R. M. Escherichia coli lipoyl synthase binds two distinct [4Fe–4S] clusters per polypeptide. Biochemistry 2004, 43, 11770– 11781,  DOI: 10.1021/bi0488505
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      Escherichia coli Lipoyl Synthase Binds Two Distinct [4Fe-4S] Clusters per Polypeptide
      Cicchillo, Robert M.; Lee, Kyung-Hoon; Baleanu-Gogonea, Camelia; Nesbitt, Natasha M.; Krebs, Carsten; Booker, Squire J.
      Biochemistry (2004), 43 (37), 11770-11781CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-L-methionine (SAM) as the precursor to a high-energy 5'-deoxyadenosyl 5'-radical (5'-dA•). In the LS reaction, the 5'-dA• is hypothesized to abstr. hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM are required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d15 H-protein of the glycine cleavage system-one of the substrates for LS-has been reported. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X3-C-X2-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X4-C-X5-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 ± 0.5 irons and 6.4 ± 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5'-deoxyadenosine (5'-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 ± 0.1 irons and 3.6 ± 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 ± 0.1 irons and 4.7 ± 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5'-dA or the lipoyl group. Moessbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all assocd. iron is in the configuration [4Fe-4S]2+. When wild-type LS was reconstituted with 57Fe and sodium sulfide, it harbored considerably more iron (13.8 ± 0.6) and sulfide (13.1 ± 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5'-dA and 0.36 equiv of the lipoyl group. Moessbauer spectroscopy of this protein revealed that only ∼67% ± 6% of the iron is in the form of [4Fe-4S]2+ clusters, amounting to 9.2 ± 0.4 irons and 8.8 ± 0.1 sulfides or 2 [4Fe-4S]2+ clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the Moessbauer parameters of the clusters assocd. with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concn. and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-contg. motifs.
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      Douglas, P. Lipoyl synthase inserts sulfur atoms into an octanoyl substrate in a stepwise manner. Angew. Chem., Int. Ed. Engl. 2006, 45 (31), 5197– 9,  DOI: 10.1002/anie.200601910
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      Lipoyl synthase inserts sulfur atoms into an octanoyl substrate in a stepwise manner
      Douglas Paul; Kriek Marco; Bryant Penny; Roach Peter L
      Angewandte Chemie (International ed. in English) (2006), 45 (31), 5197-9 ISSN:1433-7851.
      There is no expanded citation for this reference.
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      Lanz, N. D. Evidence for a catalytically and kinetically competent enzyme-substrate cross-linked intermediate in catalysis by lipoyl synthase. Biochemistry 2014, 53, 4557– 4572,  DOI: 10.1021/bi500432r
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      Evidence for a Catalytically and Kinetically Competent Enzyme-Substrate Cross-Linked Intermediate in Catalysis by Lipoyl Synthase
      Lanz, Nicholas D.; Pandelia, Maria-Eirini; Kakar, Elizabeth S.; Lee, Kyung-Hoon; Krebs, Carsten; Booker, Squire J.
      Biochemistry (2014), 53 (28), 4557-4572CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Lipoyl synthase (LS) catalyzes the final step in lipoyl cofactor biosynthesis: the insertion of two sulfur atoms at C6 and C8 of an (N6-octanoyl)-lysyl residue on a lipoyl carrier protein (LCP). LS is a member of the radical SAM superfamily, enzymes that use a [4Fe-4S] cluster to effect the reductive cleavage of S-adenosyl-L-methionine (SAM) to L-methionine and a 5'-deoxyadenosyl 5'-radical (5'-dA•). In the LS reaction, two equiv. of 5'-dA• are generated sequentially to abstr. hydrogen atoms from C6 and C8 of the appended octanoyl group, initiating sulfur insertion at these positions. The second [4Fe-4S] cluster on LS, termed the auxiliary cluster, is proposed to be the source of the inserted sulfur atoms. Herein, we provide evidence for the formation of a covalent cross-link between LS and an LCP or synthetic peptide substrate in reactions in which insertion of the second sulfur atom is slowed significantly by deuterium substitution at C8 or by inclusion of limiting concns. of SAM. The observation that the proteins elute simultaneously by anion-exchange chromatog. but are sepd. by aerobic SDS-PAGE is consistent with their linkage through the auxiliary cluster that is sacrificed during turnover. Generation of the cross-linked species with a small, unlabeled (N6-octanoyl)-lysyl-contg. peptide substrate allowed demonstration of both its chem. and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction. Mossbauer spectroscopy of the cross-linked intermediate reveals that one of the [4Fe-4S] clusters, presumably the auxiliary cluster, is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to those of reduced [3Fe-4S]0 clusters.
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      Lanz, N. D. Characterization of a radical intermediate in lipoyl cofactor biosynthesis. J. Am. Chem. Soc. 2015, 137, 13216– 13219,  DOI: 10.1021/jacs.5b04387
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      Characterization of a radical intermediate in lipoyl cofactor biosynthesis
      Lanz, Nicholas D.; Rectenwald, Justin M.; Wang, Bo; Kakar, Elizabeth S.; Laremore, Tatiana N.; Booker, Squire J.; Silakov, Alexey
      Journal of the American Chemical Society (2015), 137 (41), 13216-13219CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Lipoyl synthase (LipA) catalyzes the final step in the biosynthesis of lipoyl cofactor, the insertion of 2 sulfur atoms at C6 and C8 of an n-octanoyl chain. LipA is a member of the radical S-adenosylmethionine (SAM) superfamily of enzymes and uses 2 [4Fe-4S] clusters to catalyze its transformation. One cluster binds in contact with SAM and donates the requisite electron for the reductive cleavage of SAM to generate 2 5'-deoxyadenosyl 5'-radicals, which abstr. H atoms from C6 and C8 of the substrate. By contrast, the 2nd, auxiliary [4Fe-4S] cluster, has been hypothesized to serve as the sulfur donor in the reaction. Such a sacrificial role for an Fe-S cluster during catalysis has not been universally accepted. The use of a conjugated 2,4-hexadienoyl-contg. substrate analog has allowed the substrate radical to be trapped and characterized by continuous-wave and pulsed ESR methods. Here, the authors report the observation of a 57Fe hyperfine coupling interaction with the paramagnetic signal, which indicates that the Fe-S cluster of LipA and its substrate are within bonding distance.
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    38. 38
      Lanz, N. D.; Booker, S. J. Auxiliary iron-sulfur cofactors in radical SAM enzymes. Biochim. Biophys. Acta 2015, 1853 (6), 1316– 34,  DOI: 10.1016/j.bbamcr.2015.01.002
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      Auxiliary iron-sulfur cofactors in radical SAM enzymes
      Lanz, Nicholas D.; Booker, Squire J.
      Biochimica et Biophysica Acta, Molecular Cell Research (2015), 1853 (6), 1316-1334CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
      A review. A vast no. of enzymes are now known to belong to a superfamily known as radical SAM, which all contain a [4Fe-4S] cluster ligated by three cysteine residues. The remaining, unligated, iron ion of the cluster binds in contact with the α-amino and α-carboxylate groups of S-adenosyl-L-methionine (SAM). This binding mode facilitates inner-sphere electron transfer from the reduced form of the cluster into the sulfur atom of SAM, resulting in a reductive cleavage of SAM to methionine and a 5'-deoxyadenosyl radical. The 5'-deoxyadenosyl radical then abstrs. a target substrate hydrogen atom, initiating a wide variety of radical-based transformations. A subset of radical SAM enzymes contains one or more addnl. iron-sulfur clusters that are required for the reactions they catalyze. However, outside of a subset of sulfur insertion reactions, very little is known about the roles of these addnl. clusters. This review will highlight the most recent advances in the identification and characterization of radical SAM enzymes that harbor auxiliary iron-sulfur clusters. This article is part of a Special Issue entitled: Fe/S proteins: Anal., structure, function, biogenesis and diseases.
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    39. 39
      Lanz, N. D.; Booker, S. J. The role of iron-sulfur clusters in the biosynthesis of the lipoyl cofactor. In Iron-Sulfur Clusters in Chemistry and Biology; Rouault, T. A., Ed.; Walter de Gruyter GMbH: Berlin, Germany, 2014.
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      Lanz, N. D.; Booker, S. J. Identification and function of auxiliary iron-sulfur clusters in radical SAM enzymes. Biochim. Biophys. Acta 2012, 1824 (11), 1196– 212,  DOI: 10.1016/j.bbapap.2012.07.009
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      40
      Identification and function of auxiliary iron-sulfur clusters in radical SAM enzymes
      Lanz, Nicholas D.; Booker, Squire J.
      Biochimica et Biophysica Acta, Proteins and Proteomics (2012), 1824 (11), 1196-1212CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)
      A review. Radical SAM (RS) enzymes use a 5'-deoxyadenosyl 5'-radical generated from a reductive cleavage of S-adenosyl-L-methionine to catalyze over 40 distinct reaction types. A distinguishing feature of these enzymes is a [4Fe-4S] cluster to which each of three iron ions is ligated by three cysteinyl residues most often located in a Cx3Cx2C motif. The α-amino and α-carboxylate groups of SAM anchor the mol. to the remaining iron ion, which presumably facilitates its reductive cleavage. A subset of RS enzymes contains addnl. iron-sulfur clusters, - which we term auxiliary clusters - most of which have unidentified functions. Enzymes in this subset are involved in cofactor biosynthesis and maturation, post-transcriptional and post-translational modification, enzyme activation, and antibiotic biosynthesis. The addnl. clusters in these enzymes have been proposed to function in sulfur donation, electron transfer, and substrate anchoring. This review will highlight evidence supporting the presence of multiple iron-sulfur clusters in these enzymes as well as their predicted roles in catalysis.
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    41. 41
      Walsby, C. J. An anchoring role for FeS clusters: chelation of the amino acid moiety of S-adenosylmethionine to the unique iron site of the [4Fe–4S] cluster of pyruvate formate–lyase activating enzyme. J. Am. Chem. Soc. 2002, 124, 11270– 11271,  DOI: 10.1021/ja027078v
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      41
      An Anchoring Role for FeS Clusters: Chelation of the Amino Acid Moiety of S-Adenosylmethionine to the Unique Iron Site of the [4Fe-4S] Cluster of Pyruvate Formate-Lyase Activating Enzyme
      Walsby, Charles J.; Ortillo, Danilo; Broderick, William E.; Broderick, Joan B.; Hoffman, Brian M.
      Journal of the American Chemical Society (2002), 124 (38), 11270-11271CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Pyruvate formate-lyase activating enzyme (PFL-AE) generates the catalytically essential glycyl radical on pyruvate formate-lyase via the interaction of the catalytically active (4Fe-4S)+ cluster with S-adenosylmethionine (AdoMet). Like other members of the Fe-S/AdoMet family of enzymes, PFL-AE is thought to function via generation of an AdoMet-derived 5'-deoxyadenosyl radical intermediate; however, the mechanistic steps by which this radical is generated remain to be elucidated. While all of the members of the Fe-S/AdoMet family of enzymes appear to have a unique iron site in the (4Fe-4S) cluster, based on the presence of a conserved three-cysteine cluster binding motif, the role of this unique site has been elusive. Here we utilize 35-GHz pulsed electron nuclear double resonance (ENDOR) studies of the (4Fe-4S)+ cluster of PFL-AE in complex with isotopically labeled AdoMet [denoted (1+/AdoMet)] to show that the unique iron serves to anchor the AdoMet for catalysis. AdoMet labeled with 17O at the carboxylate shows a coupling of A = 12.2 MHz, consistent with direct coordination of the carboxylate to the unique iron of the cluster. This is supported by 13C-ENDOR with the carboxylato carbon labeled with 13C, which shows a hyperfine coupling of 0.71 MHz. AdoMet enriched with 15N at the amino position gives rise to a spectrum with A(15N) = 5.8 MHz, consistent with direct coordination of the amino group to a unique iron of the cluster. Together, the results demonstrate that the unique iron of the (4Fe-4S) cluster anchors AdoMet by forming a classical N/O chelate with the amino and carboxylato groups of the methionine fragment.
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      Walsby, C. J. Electron-nuclear double resonance spectroscopic evidence that S-adenosylmethionine binds in contact with the catalytically active [4Fe-4S]+ cluster of pyruvate formate-lyase activating enzyme. J. Am. Chem. Soc. 2002, 124, 3143– 3151,  DOI: 10.1021/ja012034s
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      Electron-Nuclear Double Resonance Spectroscopic Evidence That S-Adenosylmethionine Binds in Contact with the Catalytically Active [4Fe-4S]+ Cluster of Pyruvate Formate-Lyase Activating Enzyme
      Walsby, Charles J.; Hong, Wei; Broderick, William E.; Cheek, Jennifer; Ortillo, Danilo; Broderick, Joan B.; Hoffman, Brian M.
      Journal of the American Chemical Society (2002), 124 (12), 3143-3151CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Pyruvate formate-lyase activating enzyme (PFL-AE) is a representative member of an emerging family of enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (AdoMet) to initiate radical catalysis. Although these enzymes have diverse functions, evidence is emerging that they operate by a common mechanism in which a [4Fe-4S]+ interacts with AdoMet to generate a 5'-deoxyadenosyl radical intermediate. To date, however, it has been unclear whether the iron-sulfur cluster is a simple electron-transfer center or whether it participates directly in the radical generation chem. Here we utilize ESR (EPR) and pulsed 35 GHz electron-nuclear double resonance (ENDOR) spectroscopy to address this question. EPR spectroscopy reveals a dramatic effect of AdoMet on the EPR spectrum of the [4Fe-4S]+ of PFL-AE, changing it from rhombic (g = 2.02, 1.94, 1.88) to nearly axial (g = 2.01, 1.88, 1.87). 2H and 13C ENDOR spectroscopy was performed on [4Fe-4S]+-PFL-AE (S = 1/2) in the presence of AdoMet labeled at the Me position with either 2H or 13C (denoted [1+/AdoMet]). The observation of a substantial 2H coupling of ∼1 MHz (∼6-7 MHz for 1H), as well as hyperfine-split signals from the 13C, manifestly require that AdoMet lie close to the cluster. 2H and 13C ENDOR data were also obtained for the interaction of AdoMet with the diamagnetic [4Fe-4S]2+ state of PFL-AE, which is visualized through cryoredn. of the frozen [4Fe-4S]2+/AdoMet complex to form the reduced state (denoted [2+/AdoMet]red) trapped in the structure of the oxidized state. 2H and 13C ENDOR spectra for [2+/AdoMet]red are essentially identical to those obtained for the [1+/AdoMet] samples, showing that the cofactor binds in the same geometry to both the 1+ and 2+ states of PFL-AE. Anal. of 2D field-frequency 13C ENDOR data reveals an isotropic hyperfine contribution, which requires that AdoMet lie in contact with the cluster, weakly interacting with it through an incipient bond/antibond. From the anisotropic hyperfine contributions for the 2H and 13C ENDOR, we have estd. the distance from the closest Me proton of AdoMet to the closest iron of the cluster to be ∼3.0-3.8 Å, while the distance from the Me carbon to the nearest iron is ∼4-5 Å. We have used this information to construct a model for the interaction of AdoMet with the [4Fe-4S]2+/+ cluster of PFL-AE and have proposed a mechanism for radical generation that is consistent with these results.
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    43. 43
      Vey, J. L.; Drennan, C. L. Structural Insights into Radical Generation by the Radical SAM Superfamily. Chem. Rev. 2011, 111 (4), 2487– 2506,  DOI: 10.1021/cr9002616
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      Structural insights into radical generation by the radical SAM superfamily
      Vey, Jessica L.; Drennan, Catherine L.
      Chemical Reviews (Washington, DC, United States) (2011), 111 (4), 2487-2506CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The radical adenosylmethionine (SAM) enzymes are a newly identified enzyme superfamily capable of catalyzing radical chem. similar to, but more extensive than that performed by adenosylcobalamin (AdoCbl)-dependent enzymes. The radical SAM and AdoCbl-dependent enzymes have in common the 5'-dA· intermediate, a highly oxidizing and unstable radical intermediate that has never been directly obsd. However, its existence has been shown in both the radical SAM and AdoCbl-dependent systems. Since the classification of radical SAM enzymes as a superfamily, researchers have elucidated key details of the radical generation processes, begun characterization of new radical SAM enzymes, and published the 1st few crystal structures of superfamily members. Here, the authors focus on key aspects of the 1st series of radical SAM structures in order to highlight the structural features of the superfamily and to identify the main elements involved in substrate binding and catalysis.
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    44. 44
      Harmer, J. E. Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions. Biochem. J. 2014, 464, 123– 133,  DOI: 10.1042/BJ20140895
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      Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions
      Harmer, Jenny E.; Hiscox, Martyn J.; Dinis, Pedro C.; Fox, Stephen J.; Iliopoulos, Andreas; Hussey, James E.; Sandy, James; Van Beek, Florian T.; Essex, Jonathan W.; Roach, Peter L.
      Biochemical Journal (2014), 464 (1), 123-133CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      Lipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) enzyme superfamily. In the present study, we solved crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX4CX5C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site-directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystd. lipoyl synthase (LipA) complex contains 5'-methylthioadenosine (MTA), a breakdown product of SAM, bound in the likely SAM-binding site. Modeling has identified an 18 Å (1 Å=0.1 nm) deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest that the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enable.
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    45. 45
      McLaughlin, M. I. Crystallographic snapshots of sulfur insertion by lipoyl synthase. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (34), 9446– 50,  DOI: 10.1073/pnas.1602486113
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      45
      Crystallographic snapshots of sulfur insertion by lipoyl synthase
      McLaughlin, Martin I.; Lanz, Nicholas D.; Goldman, Peter J.; Lee, Kyung-Hoon; Booker, Squire J.; Drennan, Catherine L.
      Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (34), 9446-9450CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at the unactivated C6 and C8 positions of a protein-bound octanoyl chain to produce the lipoyl cofactor. To activate its substrate for sulfur insertion, LipA uses a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet) radical chem.; the remainder of the reaction mechanism, esp. the source of the sulfur, has been less clear. One controversial proposal involves the removal of sulfur from a second (auxiliary) [4Fe-4S] cluster on the enzyme, resulting in destruction of the cluster during each round of catalysis. Here, we present two high-resoln. crystal structures of LipA from Mycobacterium tuberculosis: one in its resting state and one at an intermediate state during turnover. In the resting state, an auxiliary [4Fe-4S] cluster has an unusual serine ligation to one of the irons. After reaction with an octanoyllysine-contg. 8-mer peptide substrate and 1 equiv AdoMet, conditions that allow for the first sulfur insertion but not the second insertion, the serine ligand dissocs. from the cluster, the iron ion is lost, and a sulfur atom that is still part of the cluster becomes covalently attached to C6 of the octanoyl substrate. This intermediate structure provides a clear picture of iron-sulfur cluster destruction in action, supporting the role of the auxiliary cluster as the sulfur source in the LipA reaction and describing a radical strategy for sulfur incorporation into completely unactivated substrates.
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    46. 46
      McCarthy, E. L. The A-type domain in Escherichia coli NfuA is required for regenerating the auxiliary [4Fe-4S] cluster in Escherichia coli lipoyl synthase. J. Biol. Chem. 2019, 294 (5), 1609– 1617,  DOI: 10.1074/jbc.RA118.006171
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      46
      The A-type domain in Escherichia coli NfuA is required for regenerating the auxiliary [4Fe-4S] cluster in Escherichia coli lipoyl synthase
      McCarthy, Erin L.; Rankin, Ananda N.; Dill, Zerick R.; Booker, Squire J.
      Journal of Biological Chemistry (2019), 294 (5), 1609-1617CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The lipoyl cofactor plays an integral role in several essential biol. processes. The last step in its de novo biosynthetic pathway, the attachment of two sulfur atoms at C6 and C8 of an n-octanoyllysyl chain, is catalyzed by lipoyl synthase (LipA), a member of the radical SAM superfamily. In addn. to the [4Fe-4S] cluster common to all radical SAM enzymes, LipA contains a second [4Fe-4S] auxiliary cluster, which is sacrificed during catalysis to supply the requisite sulfur atoms, rendering the protein inactive for further turnovers. Recently, it was shown that the Fe-S cluster carrier protein NfuA from Escherichia coli can regenerate the auxiliary cluster of E. coli LipA after each turnover, but the mol. mechanism is incompletely understood. Herein, using protein-protein interaction and kinetic assays as well as site-directed mutagenesis, we provide further insight into the mechanism of NfuA-mediated cluster regeneration. In particular, we show that the N-terminal A-type domain of E. coli NfuA is essential for its tight interaction with LipA. Further, we demonstrate that NfuA from Mycobacterium tuberculosis can also regenerate the auxiliary cluster of E. coli LipA. However, an Nfu protein from Staphylococcus aureus, which lacks the A-type domain, was severely diminished in facilitating cluster regeneration. Of note, addn. of the N-terminal domain of E. coli NfuA to S. aureus Nfu, fully restored cluster-regenerating activity. These results expand our understanding of the newly discovered mechanism by which the auxiliary cluster of LipA is restored after each turnover.
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      McCarthy, E. L.; Booker, S. J. Destruction and reformation of an iron-sulfur cluster during catalysis by lipoyl synthase. Science 2017, 358 (6361), 373– 377,  DOI: 10.1126/science.aan4574
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      Destruction and reformation of an iron-sulfur cluster during catalysis by lipoyl synthase
      McCarthy, Erin L.; Booker, Squire J.
      Science (Washington, DC, United States) (2017), 358 (6361), 373-377CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Lipoyl synthase (LipA) catalyzes the last step in the biosynthesis of the lipoyl cofactor, which is the attachment of two sulfhydryl groups to C6 and C8 of a pendant octanoyl chain. The appended sulfur atoms derive from an auxiliary [4Fe-4S] cluster on the protein that is degraded during turnover, limiting LipA to one turnover in vitro. We found that the Escherichia coli iron-sulfur (Fe-S) cluster carrier protein NfuA efficiently reconstitutes the auxiliary cluster during LipA catalysis in a step that is not rate-limiting. We also found evidence for a second pathway for cluster regeneration involving the E. coli protein IscU. These results show that enzymes that degrade their Fe-S clusters as a sulfur source can nonetheless act catalytically. Our results also explain why patients with NFU1 gene deletions exhibit phenotypes that are indicative of lipoyl cofactor deficiencies.
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      Maio, N.; Rouault, T. A. Iron-sulfur cluster biogenesis in mammalian cells: New insights into the molecular mechanisms of cluster delivery. Biochim. Biophys. Acta 2015, 1853 (6), 1493– 512,  DOI: 10.1016/j.bbamcr.2014.09.009
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      Iron -sulfur cluster biogenesis in mammalian cells: New insights into the molecular mechanisms of cluster delivery
      Maio, Nunziata; Rouault, Tracey A.
      Biochimica et Biophysica Acta, Molecular Cell Research (2015), 1853 (6), 1493-1512CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
      A review. Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorg. sulfur. The combination of the chem. reactivity of iron and sulfur, together with many variations of cluster compn., oxidn. states and protein environments, enables Fe-S clusters to participate in numerous biol. processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i.e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein-protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several crit. questions still remain, such as: what is the role of frataxin. Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload. How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer. This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of mol. features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Addnl., it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. This article is part of a Special Issue entitled: Fe/S proteins: Anal., structure, function, biogenesis and diseases.
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      Majewska, J. Binding of the Chaperone Jac1 Protein and Cysteine Desulfurase Nfs1 to the Iron-Sulfur Cluster Scaffold Isu Protein Is Mutually Exclusive. J. Biol. Chem. 2013, 288 (40), 29134– 29142,  DOI: 10.1074/jbc.M113.503524
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      Binding of the Chaperone Jac1 Protein and Cysteine Desulfurase Nfs1 to the Iron-Sulfur Cluster Scaffold Isu Protein Is Mutually Exclusive
      Majewska, Julia; Ciesielski, Szymon J.; Schilke, Brenda; Kominek, Jacek; Blenska, Anna; Delewski, Wojciech; Song, Ji-Yoon; Marszalek, Jaroslaw; Craig, Elizabeth A.; Dutkiewicz, Rafal
      Journal of Biological Chemistry (2013), 288 (40), 29134-29142CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Biogenesis of mitochondrial iron-sulfur (Fe/S) cluster proteins requires the interaction of multiple proteins with the highly conserved 14-kDa scaffold protein Isu, on which clusters are built prior to their transfer to recipient proteins. For example, the assembly process requires the cysteine desulfurase Nfs1, which serves as the sulfur donor for cluster assembly. The transfer process requires Jac1, a J-protein Hsp70 cochaperone. We recently identified three residues on the surface of Jac1 that form a hydrophobic patch crit. for interaction with Isu. The results of mol. modeling of the Isu1-Jac1 interaction, which was guided by these exptl. data and structural/biophys. information available for bacterial homologs, predicted the importance of three hydrophobic residues forming a patch on the surface of Isu1 for interaction with Jac1. Using Isu variants having alterations in residues that form the hydrophobic patch on the surface of Isu, this prediction was exptl. validated by in vitro binding assays. In addn., Nfs1 was found to require the same hydrophobic residues of Isu for binding, as does Jac1, suggesting that Jac1 and Nfs1 binding is mutually exclusive. In support of this conclusion, Jac1 and Nfs1 compete for binding to Isu. Evolutionary anal. revealed that residues involved in these interactions are conserved and that they are crit. residues for the biogenesis of Fe/S cluster protein in vivo. We propose that competition between Jac1 and Nfs1 for Isu binding plays an important role in transitioning the Fe/S cluster biogenesis machinery from the cluster assembly step to the Hsp70-mediated transfer of the Fe/S cluster to recipient proteins.
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      Vickery, L. E.; Cupp-Vickery, J. R. Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation. Crit Rev. Biochem Mol. Biol. 2007, 42 (2), 95– 111,  DOI: 10.1080/10409230701322298
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      50
      Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation
      Vickery, Larry E.; Cupp-Vickery, Jill R.
      Critical Reviews in Biochemistry and Molecular Biology (2007), 42 (2), 95-111CODEN: CRBBEJ; ISSN:1040-9238. (Informa Healthcare USA, Inc.)
      A review. Genetic and biochem. studies have led to the identification of several cellular pathways for the biosynthesis of iron-sulfur proteins in different organisms. The most broadly distributed and highly conserved system involves an Hsp70 chaperone and J-protein co-chaperone system that interacts with a scaffold-like protein involved in [FeS]-cluster preassembly. Specialized forms of Hsp70 and their co-chaperones have evolved in bacteria (HscA, HscB) and in certain fungi (Ssq1, Jac1), whereas most eukaryotes employ a multifunctional mitochondrial Hsp70 (mtHsp70) together with a specialized co-chaperone homologous to HscB/Jac1. HscA and Ssq1 have been shown to specifically bind to a conserved sequence present in the [FeS]-scaffold protein designated IscU in bacteria and Isu in fungi, and the crystal structure of a complex of a peptide contg. the IscU recognition region bound to the HscA substrate binding domain has been detd. The interaction of IscU/Isu with HscA/Ssq1 is regulated by HscB/Jac1 which bind the scaffold protein to assist delivery to the chaperone and stabilize the chaperone-scaffold complex by enhancing chaperone ATPase activity. The crystal structure of HscB reveals that the N-terminal J-domain involved in regulation of HscA ATPase activity is similar to other J-proteins, whereas the C-terminal domain is unique and appears to mediate specific interactions with IscU. At the present time the exact function(s) of chaperone-[FeS]-scaffold interactions in iron-sulfur protein biosynthesis remain(s) to be established. In vivo and in vitro studies of yeast Ssq1 and Jac1 indicate that the chaperones are not required for [FeS]-cluster assembly on Isu. Recent in vitro studies using bacterial HscA, HscB and IscU have shown that the chaperones destabilize the IscU[FeS] complex and facilitate cluster delivery to an acceptor apo-protein consistent with a role in regulating cluster release and transfer. Addnl. genetic and biochem. studies are needed to extend these findings to mtHsp70 activities in higher eukaryotes.
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      Fox, N. G. The Human Iron-Sulfur Assembly Complex Catalyzes the Synthesis of [2Fe-2S] Clusters on ISCU2 That Can. Be Transferred to Acceptor Molecules. Biochemistry 2015, 54 (25), 3871– 9,  DOI: 10.1021/bi5014485
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      The Human Iron-Sulfur Assembly Complex Catalyzes the Synthesis of [2Fe-2S] Clusters on ISCU2 That Can Be Transferred to Acceptor Molecules
      Fox, Nicholas G.; Chakrabarti, Mrinmoy; McCormick, Sean P.; Lindahl, Paul A.; Barondeau, David P.
      Biochemistry (2015), 54 (25), 3871-3879CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Iron-sulfur (Fe-S) clusters are essential protein cofactors for most life forms. In human mitochondria, the core Fe-S biosynthetic enzymic complex (called SDUF) consists of NFS1, ISD11, ISCU2, and frataxin (FXN) protein components. Few mechanistic details about how this complex synthesizes Fe-S clusters and how these clusters are delivered to targets are known. Here CD and Mossbauer spectroscopies were used to reveal details of the Fe-S cluster assembly reaction on the SDUF complex. SDUF reactions generated [2Fe-2S] cluster intermediates that readily converted to stable [2Fe-2S] clusters bound to uncomplexed ISCU2. Similar reactions that included the apo Fe-S acceptor protein human ferredoxin (FDX1) resulted in formation of [2Fe-2S]-ISCU2 rather than [2Fe-2S]-FDX1. Subsequent addn. of dithiothreitol (DTT) induced transfer of the cluster from ISCU2 to FDX1, suggesting that [2Fe-2S]-ISCU2 is an intermediate. Reactions that initially included DTT rapidly generated [2Fe-2S]-FDX1 and bypassed formation of [2Fe-2S]-ISCU2. In the absence of apo-FDX1, incubation of [2Fe-2S]-ISCU2 with DTT generated [4Fe-4S]-ISCU2 species. Together, these results conflict with a recent report of stable [4Fe-4S] cluster formation on the SDUF complex. Rather, they support a model in which SDUF builds transient [2Fe-2S] cluster intermediates that generate clusters on sulfur-contg. mols., including uncomplexed ISCU2. Addnl. small mol. or protein factors are required for the transfer of these clusters to Fe-S acceptor proteins or the synthesis of [4Fe-4S] clusters.
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      Braymer, J. J.; Lill, R. Iron-sulfur cluster biogenesis and trafficking in mitochondria. J. Biol. Chem. 2017, 292 (31), 12754– 12763,  DOI: 10.1074/jbc.R117.787101
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      Iron-sulfur cluster biogenesis and trafficking in mitochondria
      Braymer, Joseph J.; Lill, Roland
      Journal of Biological Chemistry (2017), 292 (31), 12754-12763CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      A review. The biogenesis of Fe-S proteins in eukaryotes is a multistage, multicompartment process that is essential for a broad range of cellular functions, including genome maintenance, protein translation, energy conversion, and the antiviral response. Genetic and cell biol. studies over almost 2 decades have revealed ∼30 proteins involved in the synthesis of cellular [2Fe-2S] and [4Fe-4S] clusters and their incorporation into numerous apoproteins. Mechanistic aspects of Fe-S protein biogenesis continue to be elucidated by biochem. and ultrastructural investigations. Here, we review recent developments in the pursuit of constructing a comprehensive model of Fe-S protein assembly in the mitochondrion.
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      Braymer, J. J. Mechanistic concepts of iron-sulfur protein biogenesis in Biology. Biochim Biophys Acta Mol. Cell Res. 2021, 1868 (1), 118863,  DOI: 10.1016/j.bbamcr.2020.118863
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      Mechanistic concepts of iron-sulfur protein biogenesis in Biology
      Braymer, Joseph J.; Freibert, Sven A.; Rakwalska-Bange, Magdalena; Lill, Roland
      Biochimica et Biophysica Acta, Molecular Cell Research (2021), 1868 (1), 118863CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
      A review. Iron-sulfur (Fe/S) proteins are present in virtually all living organisms and are involved in numerous cellular processes such as respiration, photosynthesis, metabolic reactions, nitrogen fixation, radical biochem., protein synthesis, antiviral defense, and genome maintenance. Their versatile functions may go back to the proposed role of their Fe/S cofactors in the origin of life as efficient catalysts and electron carriers. In prokaryotes, three Fe/S protein biogenesis systems are known; ISC, SUF, and the more specialized NIF. The former two systems have been transferred by endosymbiosis from bacteria to mitochondria and plastids, resp., of eukaryotes. In their cytosol, eukaryotes use the CIA machinery for the biogenesis of cytosolic and nuclear Fe/S proteins. Despite the structural diversity of the protein constituents of these four machineries, general mechanistic concepts underlie the complex process of Fe/S protein biogenesis. This provides a comprehensive and comparative overview of the various known biogenesis systems in Biol., and summarizes their common or diverging mol. mechanisms, thereby illustrating both the conservation and diverse adaptions of these four machineries during evolution and under different lifestyles. Knowledge of these fundamental biochem. pathways is not only of basic scientific interest, but is important for the understanding of human 'Fe/S diseases' and can be used in biotechnol.
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      Pérard, J.; Ollagnier de Choudens, S. Iron–sulfur clusters biogenesis by the SUF machinery: close to the molecular mechanism understanding. JBIC Journal of Biological Inorganic Chemistry 2018, 23 (4), 581– 596,  DOI: 10.1007/s00775-017-1527-3
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      Iron-sulfur clusters biogenesis by the SUF machinery: close to the molecular mechanism understanding
      Perard, J.; Ollagnier de Choudens, Sandrine
      JBIC, Journal of Biological Inorganic Chemistry (2018), 23 (4), 581-596CODEN: JJBCFA; ISSN:0949-8257. (Springer)
      Review. Fe-S clusters are among the most ancient and versatile inorg. cofactors in Nature which are used by proteins for fundamental biol. processes. Multiprotein machineries (NIF, ISC, SUF) exist for Fe-S cluster biogenesis which are mainly conserved from bacteria to human. The SUF system (sufABCDSE operon) plays a general role in many bacteria under conditions of Fe limitation or oxidative stress. Here, we summarize the current understanding of the mol. mechanism of Fe-S biogenesis by the SUF machinery. The advances in our understanding of the mol. aspects of SUF originate from biochem., biophys. and recent structural studies. Combined with recent in vivo expts., the understanding of the Fe-S biogenesis mechanism has considerably moved forward.
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      Rouault, T. A.; Maio, N. Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways. J. Biol. Chem. 2017, 292 (31), 12744– 12753,  DOI: 10.1074/jbc.R117.789537
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      Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways
      Rouault, Tracey A.; Maio, Nunziata
      Journal of Biological Chemistry (2017), 292 (31), 12744-12753CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      A review. Fe-S cofactors are composed of iron and inorg. sulfur in various stoichiometries. A complex assembly pathway conducts their initial synthesis and subsequent binding to recipient proteins. In this minireview, we discuss how discovery of the role of the mammalian cytosolic aconitase, known as iron regulatory protein 1 (IRP1), led to the characterization of the function of its Fe-S cluster in sensing and regulating cellular iron homeostasis. Moreover, we present an overview of recent studies that have provided insights into the mechanism of Fe-S cluster transfer to recipient Fe-S proteins.
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      Rouault, T. A. Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease. Dis Model Mech 2012, 5 (2), 155– 64,  DOI: 10.1242/dmm.009019
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      Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease
      Rouault Tracey A
      Disease models & mechanisms (2012), 5 (2), 155-64 ISSN:.
      Iron-sulfur (Fe-S) clusters are ubiquitous cofactors composed of iron and inorganic sulfur. They are required for the function of proteins involved in a wide range of activities, including electron transport in respiratory chain complexes, regulatory sensing, photosynthesis and DNA repair. The proteins involved in the biogenesis of Fe-S clusters are evolutionarily conserved from bacteria to humans, and many insights into the process of Fe-S cluster biogenesis have come from studies of model organisms, including bacteria, fungi and plants. It is now clear that several rare and seemingly dissimilar human diseases are attributable to defects in the basic process of Fe-S cluster biogenesis. Although these diseases -which include Friedreich's ataxia (FRDA), ISCU myopathy, a rare form of sideroblastic anemia, an encephalomyopathy caused by dysfunction of respiratory chain complex I and multiple mitochondrial dysfunctions syndrome - affect different tissues, a feature common to many of them is that mitochondrial iron overload develops as a secondary consequence of a defect in Fe-S cluster biogenesis. This Commentary outlines the basic steps of Fe-S cluster biogenesis as they have been defined in model organisms. In addition, it draws attention to refinements of the process that might be specific to the subcellular compartmentalization of Fe-S cluster biogenesis proteins in some eukaryotes, including mammals. Finally, it outlines several important unresolved questions in the field that, once addressed, should offer important clues into how mitochondrial iron homeostasis is regulated, and how dysfunction in Fe-S cluster biogenesis can contribute to disease.
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      Banci, L. [2Fe-2S] cluster transfer in iron–sulfur protein biogenesis. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (17), 6203,  DOI: 10.1073/pnas.1400102111
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      [2Fe-2S] cluster transfer in iron-sulfur protein biogenesis
      Banci, Lucia; Brancaccio, Diego; Ciofi-Baffoni, Simone; Del Conte, Rebecca; Gadepalli, Ravisekhar; Mikolajczyk, Maciej; Neri, Sara; Piccioli, Mario; Winkelmann, Julia
      Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (17), 6203-6208CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Monothiol glutaredoxins play a crucial role in Fe-S protein biogenesis. Essentially all of them can coordinate a [2Fe-2S] cluster and have been proposed to mediate the transfer of [2Fe-2S] clusters from scaffold proteins to target apoproteins, possibly by acting as cluster transfer proteins. The mol. basis of [2Fe-2S] cluster transfer from monothiol glutaredoxins to target proteins is a fundamental, but still unresolved, aspect to be defined in Fe/S protein biogenesis. In mitochondria, monothiol glutaredoxin 5 (GRX5) is involved in the maturation of all cellular Fe-S proteins and participates in cellular Fe regulation. Here, the authors show that the structural plasticity of the dimeric state of the [2Fe-2S] bound form of human GRX5 (holo hGRX5) is the crucial factor that allows an efficient cluster transfer to the partner proteins human ISCA1 and ISCA2 by a specific protein-protein recognition mechanism. Holo hGRX5 works as a metallochaperone preventing the [2Fe-2S] cluster to be released in soln. in the presence of physiol. concns. of glutathione and forming a transient, cluster-mediated protein-protein intermediate with 2 physiol. protein partners receiving the [2Fe-2S] cluster. The cluster transfer mechanism defined here may extend to other mitochondrial [2Fe-2S] target proteins.
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      Nasta, V. A pathway for assembling [4Fe-4S](2+) clusters in mitochondrial iron-sulfur protein biogenesis. Febs j 2020, 287 (11), 2312– 2327,  DOI: 10.1111/febs.15140
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      A pathway for assembling [4Fe-4S]2+ clusters in mitochondrial iron-sulfur protein biogenesis
      Nasta, Veronica; Suraci, Dafne; Gourdoupis, Spyridon; Ciofi-Baffoni, Simone; Banci, Lucia
      FEBS Journal (2020), 287 (11), 2312-2327CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
      During its late steps, the mitochondrial iron-sulfur cluster (ISC) assembly machinery leads to the formation of [4Fe-4S] clusters. In vivo studies revealed that several proteins are implicated in the biosynthesis and trafficking of [4Fe-4S] clusters in mitochondria. However, they do not provide a clear picture into how these proteins cooperate. Here, we showed that three late-acting components of the mitochondrial ISC assembly machinery (GLRX5, BOLA3, and NFU1) are part of a ISC assembly pathway leading to the synthesis of a [4Fe-4S]2+ cluster on NFU1. We showed that the [2Fe-2S]2+ GLRX5-BOLA3 complex transfers its cluster to monomeric apo NFU1 to form, in the presence of a reductant, a [4Fe-4S]2+ cluster bound to dimeric NFU1. The cluster formation on NFU1 does not occur with [2Fe-2S]2+ GLRX5, and thus, the [4Fe-4S] cluster assembly pathway is activated only in the presence of BOLA3. These results define NFU1 as an 'assembler' of [4Fe-4S] clusters, i.e., a protein able of converting two [2Fe-2S]2+ clusters into a [4Fe-4S]2+ cluster. Finally, we found that the [4Fe-4S]2+ cluster bound to NFU1 has a coordination site which is easily accessible to sulfur-contg. ligands, as is typically obsd. in metallochaperones. This finding supports a role for NFU1 in promoting rapid and controlled cluster-exchange reaction.
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      Baker, P. R.; Friederich, M. W.; Swanson, M. A.; Shaikh, T.; Bhattacharya, K.; Scharer, G. H.; Aicher, J.; Creadon-Swindell, G.; Geiger, E.; MacLean, K. N. Variant non ketotic hyperglycinemia is caused by mutations in LIAS, BOLA3 and the novel gene GLRX5. Brain 2014, 137 (2), 366– 379,  DOI: 10.1093/brain/awt328
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      Variant non ketotic hyperglycinemia is caused by mutations in LIAS, BOLA3 and the novel gene GLRX5
      Baker Peter R 2nd; Friederich Marisa W; Swanson Michael A; Shaikh Tamim; Bhattacharya Kaustuv; Scharer Gunter H; Aicher Joseph; Creadon-Swindell Geralyn; Geiger Elizabeth; MacLean Kenneth N; Lee Wang-Tso; Deshpande Charu; Freckmann Mary-Louise; Shih Ling-Yu; Wasserstein Melissa; Rasmussen Malene B; Lund Allan M; Procopis Peter; Cameron Jessie M; Robinson Brian H; Brown Garry K; Brown Ruth M; Compton Alison G; Dieckmann Carol L; Collard Renata; Coughlin Curtis R 2nd; Spector Elaine; Wempe Michael F; Van Hove Johan L K
      Brain : a journal of neurology (2014), 137 (Pt 2), 366-79 ISSN:.
      Patients with nonketotic hyperglycinemia and deficient glycine cleavage enzyme activity, but without mutations in AMT, GLDC or GCSH, the genes encoding its constituent proteins, constitute a clinical group which we call 'variant nonketotic hyperglycinemia'. We hypothesize that in some patients the aetiology involves genetic mutations that result in a deficiency of the cofactor lipoate, and sequenced genes involved in lipoate synthesis and iron-sulphur cluster biogenesis. Of 11 individuals identified with variant nonketotic hyperglycinemia, we were able to determine the genetic aetiology in eight patients and delineate the clinical and biochemical phenotypes. Mutations were identified in the genes for lipoate synthase (LIAS), BolA type 3 (BOLA3), and a novel gene glutaredoxin 5 (GLRX5). Patients with GLRX5-associated variant nonketotic hyperglycinemia had normal development with childhood-onset spastic paraplegia, spinal lesion, and optic atrophy. Clinical features of BOLA3-associated variant nonketotic hyperglycinemia include severe neurodegeneration after a period of normal development. Additional features include leukodystrophy, cardiomyopathy and optic atrophy. Patients with lipoate synthase-deficient variant nonketotic hyperglycinemia varied in severity from mild static encephalopathy to Leigh disease and cortical involvement. All patients had high serum and borderline elevated cerebrospinal fluid glycine and cerebrospinal fluid:plasma glycine ratio, and deficient glycine cleavage enzyme activity. They had low pyruvate dehydrogenase enzyme activity but most did not have lactic acidosis. Patients were deficient in lipoylation of mitochondrial proteins. There were minimal and inconsistent changes in cellular iron handling, and respiratory chain activity was unaffected. Identified mutations were phylogenetically conserved, and transfection with native genes corrected the biochemical deficiency proving pathogenicity. Treatments of cells with lipoate and with mitochondrially-targeted lipoate were unsuccessful at correcting the deficiency. The recognition of variant nonketotic hyperglycinemia is important for physicians evaluating patients with abnormalities in glycine as this will affect the genetic causation and genetic counselling, and provide prognostic information on the expected phenotypic course.
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      Maio, N.; Jain, A.; Rouault, T. A. Mammalian iron-sulfur cluster biogenesis: Recent insights into the roles of frataxin, acyl carrier protein and ATPase-mediated transfer to recipient proteins. Curr. Opin Chem. Biol. 2020, 55, 34– 44,  DOI: 10.1016/j.cbpa.2019.11.014
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      Mammalian iron-sulfur cluster biogenesis: Recent insights into the roles of frataxin, acyl carrier protein and ATPase-mediated transfer to recipient proteins
      Maio, Nunziata; Jain, Anshika; Rouault, Tracey A.
      Current Opinion in Chemical Biology (2020), 55 (), 34-44CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      A review. The recently solved crystal structures of the human cysteine desulfurase NFS1, in complex with the LYR protein ISD11, the acyl carrier protein ACP, and the main scaffold ISCU, have shed light on the mol. interactions that govern initial cluster assembly on ISCU. Here, we aim to highlight recent insights into iron-sulfur (Fe-S) cluster (ISC) biogenesis in mammalian cells that have arisen from the crystal structures of the core ISC assembly complex. We will also discuss how ISCs are delivered to recipient proteins and the challenges that remain in dissecting the pathways that deliver clusters to numerous Fe-S recipient proteins in both the mitochondrial matrix and cytosolic compartments of mammalian cells.
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      Ahting, U.; Mayr, J. A.; Vanlander, A. V.; Hardy, S. A.; Santra, S.; Makowski, C.; Alston, C. L.; Zimmermann, F. A.; Abela, L.; Plecko, B. Clinical, biochemical, and genetic spectrum of seven patients with NFU1 deficiency. Front. Genet. 2015, 6, 123,  DOI: 10.3389/fgene.2015.00123
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      Clinical, biochemical, and genetic spectrum of seven patients with NFU1 deficiency
      Ahting Uwe; Mayr Johannes A; Zimmermann Franz A; Sperl Wolfgang; Vanlander Arnaud V; Smet Joel; Van Coster Rudy; Hardy Steven A; Alston Charlotte L; Taylor Robert W; Santra Saikat; Makowski Christine; Abela Lucia; Plecko Barbara; Rohrbach Marianne; Spranger Stephanie; Seneca Sara; Rolinski Boris; Hagendorff Angela; Hempel Maja; Meitinger Thomas; Prokisch Holger; Haack Tobias B; Freisinger Peter
      Frontiers in genetics (2015), 6 (), 123 ISSN:1664-8021.
      Disorders of the mitochondrial energy metabolism are clinically and genetically heterogeneous. An increasingly recognized subgroup is caused by defective mitochondrial iron-sulfur (Fe-S) cluster biosynthesis, with defects in 13 genes being linked to human disease to date. Mutations in three of them, NFU1, BOLA3, and IBA57, affect the assembly of mitochondrial [4Fe-4S] proteins leading to an impairment of diverse mitochondrial metabolic pathways and ATP production. Patients with defects in these three genes present with lactic acidosis, hyperglycinemia, and reduced activities of respiratory chain complexes I and II, the four lipoic acid-dependent 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). To date, five different NFU1 pathogenic variants have been reported in 15 patients from 12 families. We report on seven new patients from five families carrying compound heterozygous or homozygous pathogenic NFU1 mutations identified by candidate gene screening and exome sequencing. Six out of eight different disease alleles were novel and functional studies were performed to support the pathogenicity of five of them. Characteristic clinical features included fatal infantile encephalopathy and pulmonary hypertension leading to death within the first 6 months of life in six out of seven patients. Laboratory investigations revealed combined defects of pyruvate dehydrogenase complex (five out of five) and respiratory chain complexes I and II+III (four out of five) in skeletal muscle and/or cultured skin fibroblasts as well as increased lactate (five out of six) and glycine concentration (seven out of seven). Our study contributes to a better definition of the phenotypic spectrum associated with NFU1 mutations and to the diagnostic workup of future patients.
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      Cameron, J. Mutations in Iron-Sulfur Cluster Scaffold Genes NFU1 and BOLA3 Cause a Fatal Deficiency of Multiple Respiratory Chain and 2-Oxoacid Dehydrogenase Enzymes. American journal of human genetics 2011, 89, 486– 95,  DOI: 10.1016/j.ajhg.2011.08.011
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      Mutations in Iron-Sulfur Cluster Scaffold Genes NFU1 and BOLA3 Cause a Fatal Deficiency of Multiple Respiratory Chain and 2-Oxoacid Dehydrogenase Enzymes
      Cameron, Jessie M.; Janer, Alexandre; Levandovskiy, Valeriy; MacKay, Nevena; Rouault, Tracey A.; Tong, Wing-Hang; Ogilvie, Isla; Shoubridge, Eric A.; Robinson, Brian H.
      American Journal of Human Genetics (2011), 89 (4), 486-495CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
      Severe combined deficiency of the 2-oxoacid dehydrogenases, assocd. with a defect in lipoate synthesis and accompanied by defects in complexes I, II, and III of the mitochondrial respiratory chain, is a rare autosomal recessive syndrome with no obvious causative gene defect. A candidate locus for this syndrome was mapped to chromosomal region 2p14 by microcell-mediated chromosome transfer in two unrelated families. Unexpectedly, anal. of genes in this area identified mutations in two different genes, both of which are involved in [Fe-S] cluster biogenesis. A homozygous missense mutation, c.545G>A, near the splice donor of exon 6 in NFU1 predicting a p.Arg182Gln substitution was found in one of the families. The mutation results in abnormal mRNA splicing of exon 6, and no mature protein could be detected in fibroblast mitochondria. A single base-pair duplication c.123dupA was identified in BOLA3 in the second family, causing a frame shift that produces a premature stop codon (p.Glu42Argfs*13). Transduction of fibroblast lines with retroviral vectors expressing the mitochondrial, but not the cytosolic isoform of NFU1 and with isoform 1, but not isoform 2 of BOLA3 restored both respiratory chain function and oxoacid dehydrogenase complexes. NFU1 was previously proposed to be an alternative scaffold to ISCU for the biogenesis of [Fe-S] centers in mitochondria, and the function of BOLA3 was previously unknown. Our results demonstrate that both play essential roles in the prodn. of [Fe-S] centers for the normal maturation of lipoate-contg. 2-oxoacid dehydrogenases, and for the assembly of the respiratory chain complexes.
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      Navarro-Sastre, A. A fatal mitochondrial disease is associated with defective NFU1 function in the maturation of a subset of mitochondrial Fe-S proteins. Am. J. Hum. Genet. 2011, 89 (5), 656– 67,  DOI: 10.1016/j.ajhg.2011.10.005
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      A Fatal Mitochondrial Disease Is Associated with Defective NFU1 Function in the Maturation of a Subset of Mitochondrial Fe-S Proteins
      Navarro-Sastre, Aleix; Tort, Frederic; Stehling, Oliver; Uzarska, Marta A.; Arranz, Jose Antonio; del Toro, Mireia; Labayru, M. Teresa; Landa, Joseba; Font, Aida; Garcia-Villoria, Judit; Merinero, Begona; Ugarte, Magdalena; Gutierrez-Solana, Luis Gonzalez; Campistol, Jaume; Garcia-Cazorla, Angels; Vaquerizo, Julian; Riudor, Encarnacio; Briones, Paz; Elpeleg, Orly; Ribes, Antonia; Lill, Roland
      American Journal of Human Genetics (2011), 89 (5), 656-667CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
      We report on ten individuals with a fatal infantile encephalopathy and/or pulmonary hypertension, leading to death before the age of 15 mo. Hyperglycinemia and lactic acidosis were common findings. Glycine cleavage system and pyruvate dehydrogenase complex (PDHC) activities were low. Homozygosity mapping revealed a perfectly overlapping homozygous region of 1.24 Mb corresponding to chromosome 2 and led to the identification of a homozygous missense mutation (c.622G>T) in NFU1, which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. Nine individuals were homozygous for this mutation, whereas one was compd. heterozygous for this and a splice-site (c.545+5G>A) mutation. The biochem. phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS). Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases. Upon depletion of NFU1 by RNA interference in human cell culture, LAS and, in turn, PDHC activities were largely diminished. In addn., the amt. of succinate dehydrogenase, but no other Fe-S proteins, was decreased. In contrast, depletion of the general Fe-S scaffold protein ISCU severely affected assembly of all tested Fe-S proteins, suggesting that NFU1 performs a specific function in mitochondrial Fe-S cluster maturation. Similar biochem. effects were obsd. in Saccharomyces cerevisiae upon deletion of NFU1, resulting in lower lipoylation and SDH activity. Importantly, yeast Nfu1 protein carrying the individuals' missense mutation was functionally impaired. We conclude that NFU1 functions as a late-acting maturation factor for a subset of mitochondrial Fe-S proteins.
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      Wachnowsky, C. Understanding the Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1)-Impact of a Disease-Causing Gly208Cys Substitution on Structure and Activity of NFU1 in the Fe/S Cluster Biosynthetic Pathway. J. Mol. Biol. 2017, 429 (6), 790– 807,  DOI: 10.1016/j.jmb.2017.01.021
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      Understanding the molecular basis of multiple mitochondrial dysfunctions syndrome 1 (MMDS1)-Impact of a disease-causing Gly208Cys substitution on structure and activity of NFU1 in the Fe/S Cluster Biosynthetic Pathway
      Wachnowsky, Christine; Wesley, Nathaniel A.; Fidai, Insiya; Cowan, J. A.
      Journal of Molecular Biology (2017), 429 (6), 790-807CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Iron-sulfur (Fe/S)-cluster-contg. proteins constitute one of the largest protein classes, with varied functions that include electron transport, regulation of gene expression, substrate binding and activation, and radical generation. Consequently, the biosynthetic machinery for Fe/S clusters is evolutionarily conserved, and mutations in a variety of putative intermediate Fe/S cluster scaffold proteins can cause disease states, including multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalomyopathy. Herein, we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (Gly208Cys) near the active site of NFU1, an Fe/S scaffold protein, via an in vitro investigation into the structural and functional consequences. Anal. of protein stability and oligomeric state demonstrates that the mutant increases the propensity to dimerize and perturbs the secondary structure compn. These changes appear to underlie the severely decreased ability of mutant NFU1 to accept an Fe/S cluster from physiol. relevant sources. Therefore, the point mutation on NFU1 impairs downstream cluster trafficking and results in the disease phenotype, because there does not appear to be an alternative in vivo reconstitution path, most likely due to greater protein oligomerization from a minor structural change.
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    65. 65
      Melber, A.; Na, U.; Vashisht, A.; Weiler, B. D; Lill, R.; Wohlschlegel, J. A; Winge, D. R Role of Nfu1 and Bol3 in iron-sulfur cluster transfer to mitochondrial clients. Elife 2016, 5, e15991,  DOI: 10.7554/eLife.15991
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      65
      Role of Nfu1 and Bol3 in iron-sulfur cluster transfer to mitochondrial clients
      Melber, Andrew; Na, Un; Vashisht, Ajay; Weiler, Benjamin D.; Lill, Roland; Wohlschlege, James A.; Winge, Dennis R.
      eLife (2016), 5 (), e15991/1-e15991/24CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      Iron-sulfur (Fe-S) clusters are essential for many cellular processes, ranging from aerobic respiration, metabolite biosynthesis, ribosome assembly and DNA repair. Mutations in NFU1 and BOLA3 have been linked to genetic diseases with defects in mitochondrial Fe-S centers. Through genetic studies in yeast, we demonstrate that Nfu1 functions in a late step of [4Fe-4S] cluster biogenesis that is of heightened importance during oxidative metab. Proteomic studies revealed Nfu1 phys. interacts with components of the ISA [4Fe-4S] assembly complex and client proteins that need [4Fe-4S] clusters to function. Addnl. studies focused on the mitochondrial BolA proteins, Bol1 and Bol3 (yeast homolog to human BOLA3), revealing that Bol1 functions earlier in Fe-S biogenesis with the monothiol glutaredoxin, Grx5, and Bol3 functions late with Nfu1. Given these observations, we propose that Nfu1, assisted by Bol3, functions to facilitate Fe-S transfer from the biosynthetic app. to the client proteins preventing oxidative damage to [4Fe-4S] clusters.
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    66. 66
      Zhao, S. Assembly of the covalent linkage between lipoic acid and its cognate enzymes. Chem. Biol. 2003, 10, 1293– 1302,  DOI: 10.1016/j.chembiol.2003.11.016
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      Assembly of the Covalent Linkage between Lipoic Acid and Its Cognate Enzymes
      Zhao, Xin; Miller, J. Richard; Jiang, Yanfang; Marletta, Michael A.; Cronan, John E.
      Chemistry & Biology (2003), 10 (12), 1293-1302CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
      Lipoic acid is synthesized from octanoic acid by insertion of sulfur atoms at carbons 6 and 8 and is covalently attached to a pyruvate dehydrogenase (PDH) subunit. We show that sulfur atoms can be inserted into octanoyl moieties attached to a PDH subunit or a derived domain. Escherichia coli lipB mutants grew well when supplemented with octanoate in place of lipoate. Octanoate growth required both lipoate protein ligase (LplA) and LipA, the sulfur insertion protein, suggesting that LplA attached octanoate to the dehydrogenase and LipA then converted the octanoate to lipoate. This pathway was tested by labeling a PDH domain with deuterated octanoate in an E. coli strain devoid of LipA activity. The labeled octanoyl domain was converted to lipoylated domain upon restoration of LipA. Moreover, octanoyl domain and octanoyl-PDH were substrates for sulfur insertion in vitro.
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      Billgren, E. S.; Cicchillo, R. M.; Nesbitt, N. M.; Booker, S. J. Lipoic acid biosynthesis and enzymology. In Comprehensive Natural Products II Chemistry and Biology; Mander, L., Liu, H.-W., Eds.; Elsevier: Oxford, U.K., 2010; pp 181– 212.
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      Cicchillo, R. M.; Booker, S. J. Mechanistic investigations of lipoic acid biosynthesis in Escherichia coli: both sulfur atoms in lipoic acid are contributed by the same lipoyl synthase polypeptide. J. Am. Chem. Soc. 2005, 127, 2860– 2861,  DOI: 10.1021/ja042428u
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      68
      Mechanistic Investigations of Lipoic Acid Biosynthesis in Escherichia coli: Both Sulfur Atoms in Lipoic Acid are Contributed by the Same Lipoyl Synthase Polypeptide
      Cicchillo, Robert M.; Booker, Squire J.
      Journal of the American Chemical Society (2005), 127 (9), 2860-2861CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Lipoyl synthase catalyzes the final step in the de novo biosynthesis of the lipoyl cofactor, which is the insertion of two sulfur atoms into an octanoyl chain that is bound in an amide linkage to a conserved lysine on a lipoyl-accepting protein. We show herein that the sulfur atoms in the lipoyl cofactor are derived from lipoyl synthase itself, and that each lipoyl synthase polypeptide contributes both of the sulfur atoms to the intact cofactor.
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      Cicchillo, R. M. Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid. Biochemistry 2004, 43, 6378– 6386,  DOI: 10.1021/bi049528x
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      Lipoyl Synthase Requires Two Equivalents of S-Adenosyl-L-methionine To Synthesize One Equivalent of Lipoic Acid
      Cicchillo, Robert M.; Iwig, David F.; Jones, A. Daniel; Nesbitt, Natasha M.; Baleanu-Gogonea, Camelia; Souder, Matthew G.; Tu, Loretta; Booker, Squire J.
      Biochemistry (2004), 43 (21), 6378-6386CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various α-keto acids, as well as the cleavage of glycine into CO2 and NH3, with concomitant transfer of its α-carbon to tetrahydrofolate, generating N5,N10-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochem. studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-L-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA•), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming L-methionine in addn. to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated deriv. of the lipoyl-bearing subunit (E2) of the pyruvate dehydrogenase complex. Herein, the authors show that the octanoylated deriv. of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, the authors show that the 5'-dA• acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d15]H-protein to 5'-deoxyadenosine. Last, the authors' data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.
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      Douglas, P. Lipoyl synthase inserts sulfur atoms into an octanoyl substrate in a stepwise manner. Angew. Chem. 2006, 118, 5321– 5323,  DOI: 10.1002/ange.200601910
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      McLaughlin, M. I. Crystallographic snapshots of sulfur insertion by lipoyl synthase. Proc. Natl. Acad. Sci. U S A 2016, 113, 9446– 9450,  DOI: 10.1073/pnas.1602486113
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      Crystallographic snapshots of sulfur insertion by lipoyl synthase
      McLaughlin, Martin I.; Lanz, Nicholas D.; Goldman, Peter J.; Lee, Kyung-Hoon; Booker, Squire J.; Drennan, Catherine L.
      Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (34), 9446-9450CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at the unactivated C6 and C8 positions of a protein-bound octanoyl chain to produce the lipoyl cofactor. To activate its substrate for sulfur insertion, LipA uses a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet) radical chem.; the remainder of the reaction mechanism, esp. the source of the sulfur, has been less clear. One controversial proposal involves the removal of sulfur from a second (auxiliary) [4Fe-4S] cluster on the enzyme, resulting in destruction of the cluster during each round of catalysis. Here, we present two high-resoln. crystal structures of LipA from Mycobacterium tuberculosis: one in its resting state and one at an intermediate state during turnover. In the resting state, an auxiliary [4Fe-4S] cluster has an unusual serine ligation to one of the irons. After reaction with an octanoyllysine-contg. 8-mer peptide substrate and 1 equiv AdoMet, conditions that allow for the first sulfur insertion but not the second insertion, the serine ligand dissocs. from the cluster, the iron ion is lost, and a sulfur atom that is still part of the cluster becomes covalently attached to C6 of the octanoyl substrate. This intermediate structure provides a clear picture of iron-sulfur cluster destruction in action, supporting the role of the auxiliary cluster as the sulfur source in the LipA reaction and describing a radical strategy for sulfur incorporation into completely unactivated substrates.
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      Lanz, N. D. Characterization of Lipoyl Synthase from Mycobacterium tuberculosis. Biochemistry 2016, 55 (9), 1372– 83,  DOI: 10.1021/acs.biochem.5b01216
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      Characterization of Lipoyl Synthase from Mycobacterium tuberculosis
      Lanz, Nicholas D.; Lee, Kyung-Hoon; Horstmann, Abigail K.; Pandelia, Maria-Eirini; Cicchillo, Robert M.; Krebs, Carsten; Booker, Squire J.
      Biochemistry (2016), 55 (9), 1372-1383CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The prevalence of multiple and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is on the rise, necessitating the identification of new targets to combat an organism that has infected one-third of the world's population, according to the World Health Organization. The biosynthesis of the lipoyl cofactor is one possible target, given its crit. importance in cellular metab. and the apparent lack of functional salvage pathways in Mtb that are found in humans and many other organisms. The lipoyl cofactor is synthesized de novo in two committed steps, involving the LipB-catalyzed transfer of an octanoyl chain derived from fatty acid biosynthesis to a lipoyl carrier protein and the LipA-catalyzed insertion of sulfur atoms at C6 and C8 of the octanoyl chain. A no. of in vitro studies of lipoyl synthases from Escherichia coli, Sulfolobus solfataricus, and Thermosynechococcus elongatus have been conducted, but the enzyme from Mtb has not been characterized. Herein, we show that LipA from Mtb contains two [4Fe-4S] clusters and converts an octanoyl peptide substrate to the corresponding lipoyl peptide product via the same C6-monothiolated intermediate as that obsd. in the E. coli LipA reaction. In addn., we show that LipA from Mtb forms a complex with the H protein of the glycine cleavage system and that the strength of assocn. is dependent on the presence of S-adenosyl-L-methionine. We also show that LipA from Mtb can complement a lipA mutant of E. coli, demonstrating the commonalities of the two enzymes. Lastly, we show that the substrate for LipA, which normally acts on a post-translationally modified protein, can be reduced to carboxybenzyl-octanoyllysine.
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    73. 73
      Camponeschi, F. Paramagnetic (1)H NMR Spectroscopy to Investigate the Catalytic Mechanism of Radical S-Adenosylmethionine Enzymes. J. Mol. Biol. 2019, 431 (22), 4514– 4522,  DOI: 10.1016/j.jmb.2019.08.018
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      73
      Paramagnetic 1H NMR Spectroscopy to Investigate the Catalytic Mechanism of Radical S-Adenosylmethionine Enzymes
      Camponeschi, Francesca; Muzzioli, Riccardo; Ciofi-Baffoni, Simone; Piccioli, Mario; Banci, Lucia
      Journal of Molecular Biology (2019), 431 (22), 4514-4522CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Iron-sulfur clusters in radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chem. challenging reactions across all domains of life. Here we showed that 1H NMR spectroscopy expts. tailored to reveal hyperfine-shifted signals of metal-ligands is a powerful tool to monitor the binding of SAM and of the octanoyl-peptide substrate to the two [4Fe-4S] clusters of human lipoyl synthase. The paramagnetically shifted signals of the iron-ligands were specifically assigned to each of the two bound [4Fe-4S] clusters, and then used to examine the interaction of SAM and substrate mols. with each of the two [4Fe-4S] clusters of human lipoyl synthase. 1H NMR spectroscopy can therefore contribute to the description of the catalityc mechanism of radical SAM enzymes.
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    74. 74
      Hendricks, A. L. Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly. Int. J. Mol. Sci. 2021, 22 (4), 1598,  DOI: 10.3390/ijms22041598
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      Characterization and reconstitution of human lipoyl synthase (LIAS) supports ISCA2 and ISCU as primary cluster donors and an ordered mechanism of cluster assembly
      Hendricks, Amber L.; Wachnowsky, Christine; Fries, Brian; Fidai, Insiya; Cowan, James A.
      International Journal of Molecular Sciences (2021), 22 (4), 1598CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      Lipoyl synthase (LIAS) is an iron-sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe-4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, resp. Most information concerning LIAS and its mechanism has been detd. from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron-sulfur (Fe-S) cluster reconstitution mechanisms. Cluster donation by a no. of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe-2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liq. chromatog.-mass spectrometry (LC-MS) assay. ESR (EPR) studies of native LIAS and substituted derivs. that lacked the ability to bind one or the other of LIAS's two [4Fe-4S] clusters revealed a likely order of cluster addn., with the auxiliary cluster preceding the reducing [4Fe-4S] center. These results detail the trafficking of Fe-S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe-S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe-4S] cluster reconstitution is evident.
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      Jain, A. Assembly of the [4Fe-4S] cluster of NFU1 requires the coordinated donation of two [2Fe-2S] clusters from the scaffold proteins, ISCU2 and ISCA1. Hum. Mol. Genet. 2020, 29 (19), 3165– 3182,  DOI: 10.1093/hmg/ddaa172
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      Assembly of the [4Fe-4S] cluster of NFU1 requires the coordinated donation of two [2Fe-2S] clusters from the scaffold proteins, ISCU2 and ISCA1
      Jain, Anshika; Singh, Anamika; Maio, Nunziata; Rouault, Tracey A.
      Human Molecular Genetics (2020), 29 (19), 3165-3182CODEN: HMGEE5; ISSN:1460-2083. (Oxford University Press)
      NFU1, a late-acting iron-sulfur (Fe-S) cluster carrier protein, has a key role in the pathogenesis of the disease, multiple mitochondrial dysfunctions syndrome. In this work, using genetic and biochem. approaches, we identified the initial scaffold protein, mitochondrial ISCU (ISCU2) and the secondary carrier, ISCA1, as the direct donors of Fe-S clusters to mitochondrial NFU1, which appears to dimerize and reductively mediate the formation of a bridging [4Fe-4S] cluster, aided by ferredoxin 2. By monitoring the abundance of target proteins that acquire their Fe-S clusters from NFU1, we characterized the effects of several novel pathogenic NFU1 mutations. We obsd. that NFU1 directly interacts with each of the Fe-S cluster scaffold proteins known to ligate [2Fe-2S] clusters, ISCU2 and ISCA1, and we mapped the site of interaction to a conserved hydrophobic patch of residues situated at the end of the C-terminal alpha-helix of NFU1. Furthermore, we showed that NFU1 lost its ability to acquire its Fe-S cluster when mutagenized at the identified site of interaction with ISCU2 and ISCA1, which thereby adversely affected biochem. functions of proteins that are thought to acquire their Fe-S clusters directly from NFU1, such as lipoic acid synthase, which supports the Fe-S-dependent process of lipoylation of components of multiple key enzyme complexes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and the glycine cleavage complex.
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      Johnson, D. C.; Unciuleac, M.-C.; Dean, D. R. Controlled Expression and Functional Analysis of Iron-Sulfur Cluster Biosynthetic Components within Azotobacter vinelandii. J. Bacteriol. 2006, 188 (21), 7551– 7561,  DOI: 10.1128/JB.00596-06
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      Controlled expression and functional analysis of iron-sulfur cluster biosynthetic components within Azotobacter vinelandii
      Johnson, Deborah C.; Unciuleac, Mihaela-Carmen; Dean, Dennis R.
      Journal of Bacteriology (2006), 188 (21), 7551-7561CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      A system for the controlled expression of genes in A. vinelandii by using genomic fusions to the sucrose catabolic regulon was developed. This system was used for the functional anal. of the A. vinelandii isc genes, whose products are involved in the maturation of [Fe-S] proteins. For this anal., the scrX gene, contained within the sucrose catabolic regulon, was replaced by the contiguous A. vinelandii iscS, iscU, iscA, hscB, hscA, fdx, and iscX genes, resulting in duplicate genomic copies of these genes: one whose expression is directed by the normal isc regulatory elements (Pisc) and the other whose expression is directed by the scrX promoter (PscrX). Functional anal. of [Fe-S] protein maturation components was achieved by placing a mutation within a particular Pisc-controlled gene with subsequent repression of the corresponding PscrX-controlled component by growth on glucose as the carbon source. This exptl. strategy was used to show that IscS, IscU, HscBA, and Fdx are essential in A. vinelandii and that their depletion results in a deficiency in the maturation of aconitase, an enzyme that requires a [4Fe-4S] cluster for its catalytic activity. Depletion of IscA results in a null growth phenotype only when cells are cultured under conditions of elevated oxygen, marking the 1st null phenotype assocd. with the loss of a bacterial IscA-type protein. Furthermore, the null growth phenotype of cells depleted of HscBA could be partially reversed by culturing cells under conditions of low O2. Conserved amino acid residues within IscS, IscU, and IscA that are essential for their resp. functions and/or whose replacement results in a partial or complete dominant-neg. growth phenotype were also identified using this system.
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      Pandelia, M. E. Mössbauer spectroscopy of Fe/S proteins. Biochim. Biophys. Acta - Molecular Cell Research 2015, 1853 (6), 1395– 1405,  DOI: 10.1016/j.bbamcr.2014.12.005
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      M.ovrddot.ossbauer spectroscopy of Fe/S proteins
      Pandelia, Maria-Eirini; Lanz, Nicholas D.; Booker, Squire J.; Krebs, Carsten
      Biochimica et Biophysica Acta, Molecular Cell Research (2015), 1853 (6), 1395-1405CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
      A review. Iron-sulfur (Fe/S) clusters are structurally and functionally diverse cofactors that are found in all domains of life. 57Fe Mossbauer spectroscopy is a technique that provides information about the chem. nature of all chem. distinct Fe species contained in a sample, such as Fe oxidn. and spin state, nuclearity of a cluster with more than one metal ion, electron spin ground state of the cluster, and delocalization properties in mixed-valent clusters. Moreover, the technique allows for quantitation of all Fe species, when it is used in conjunction with ESR (EPR) spectroscopy and anal. methods. 57Fe-Mossbauer spectroscopy played a pivotal role in unraveling the electronic structures of the "well-established" [2Fe-2S]2+/+, [3Fe-4S]1+/0, and [4Fe-4S]3+/2+/1+/0 clusters and -more-recently- was used to characterize novel Fe/S clustsers, including the [4Fe-3S] cluster of the O2-tolerant hydrogenase from Aquifex aeolicus and the 3Fe-cluster intermediate obsd. during the reaction of lipoyl synthase, a member of the radical SAM enzyme superfamily.
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      Cai, K. Structural/Functional Properties of Human NFU1, an Intermediate [4Fe-4S] Carrier in Human Mitochondrial Iron-Sulfur Cluster Biogenesis. Structure 2016, 24 (12), 2080– 2091,  DOI: 10.1016/j.str.2016.08.020
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      Structural/Functional Properties of Human NFU1, an Intermediate [4Fe-4S] Carrier in Human Mitochondrial Iron-Sulfur Cluster Biogenesis
      Cai, Kai; Liu, Gaohua; Frederick, Ronnie O.; Xiao, Rong; Montelione, Gaetano T.; Markley, John L.
      Structure (Oxford, United Kingdom) (2016), 24 (12), 2080-2091CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
      Human mitochondrial NFU1 functions in the maturation of iron-sulfur proteins, and NFU1 deficiency is assocd. with a fatal mitochondrial disease. We detd. three-dimensional structures of the N- and C-terminal domains of human NFU1 by NMR spectroscopy and used these structures along with small-angle X-ray scattering (SAXS) data to derive structural models for full-length monomeric apo-NFU1, dimeric apo-NFU1 (an artifact of intermol. disulfide bond formation), and holo-NFUI (the [4Fe-4S] cluster-contg. form of the protein). Apo-NFU1 contains two cysteine residues in its C-terminal domain, and two apo-NFU1 subunits coordinate one [4Fe-4S] cluster to form a cluster-linked dimer. Holo-NFU1 consists of a complex of three of these dimers as shown by mol. wt. ests. from SAXS and size-exclusion chromatog. The SAXS-derived structural model indicates that one N-terminal region from each of the three dimers forms a tripartite interface. The activity of the holo-NFU1 prepn. was verified by demonstrating its ability to activate apo-aconitase.
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      Wachnowsky, C. Regulation of human Nfu activity in Fe-S cluster delivery-characterization of the interaction between Nfu and the HSPA9/Hsc20 chaperone complex. Febs j 2018, 285 (2), 391– 410,  DOI: 10.1111/febs.14353
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      Regulation of human Nfu activity in Fe-S cluster delivery - characterization of the interaction between Nfu and the HSPA9/Hsc20 chaperone complex
      Wachnowsky, Christine; Liu, Yushi; Yoon, Taejin; Cowan, J. A.
      FEBS Journal (2018), 285 (2), 391-410CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
      Iron-sulfur cluster biogenesis is a complex, but highly regulated process that involves de novo cluster formation from iron and sulfide ions on a scaffold protein, and subsequent delivery to final targets via a series of Fe-S cluster-binding carrier proteins. The process of cluster release from the scaffold/carrier for transfer to the target proteins may be mediated by a dedicated Fe-S cluster chaperone system. In human cells, the chaperones include heat shock protein HSPA9 and the J-type chaperone Hsc20. While the role of chaperones has been somewhat clarified in yeast and bacterial systems, many questions remain over their functional roles in cluster delivery and interactions with a variety of human Fe-S cluster proteins. One such protein, Nfu, has recently been recognized as a potential interaction partner of the chaperone complex. Herein, we examd. the ability of human Nfu to function as a carrier by interacting with the human chaperone complex. Human Nfu is shown to bind to both chaperone proteins with binding affinities similar to those obsd. for IscU binding to the homologous HSPA9 and Hsc20, while Nfu can also stimulate the ATPase activity of HSPA9. Addnl., the chaperone complex was able to promote Nfu function by enhancing the second-order rate consts. for Fe-S cluster transfer to target proteins and providing directionality in cluster transfer from Nfu by eliminating promiscuous transfer reactions. Together, these data support a hypothesis in which Nfu can serve as an alternative carrier protein for chaperone-mediated cluster release and delivery in Fe-S cluster biogenesis and trafficking.
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    80. 80
      Wesley, N. A.; Wachnowsky, C.; Fidai, I.; Cowan, J. A. Understanding the molecular basis for multiple mitochondrial dysfunctions syndrome 1 (MMDS1): impact of a disease-causing Gly189Arg substitution on NFU1. FEBS J. 2017, 284, 3838– 3848,  DOI: 10.1111/febs.14271
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      Understanding the molecular basis for multiple mitochondrial dysfunctions syndrome 1 (MMDS1): impact of a disease-causing Gly189Arg substitution on NFU1
      Wesley, Nathaniel A.; Wachnowsky, Christine; Fidai, Insiya; Cowan, J. A.
      FEBS Journal (2017), 284 (22), 3838-3848CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
      Iron-sulfur (Fe/S) cluster-contg. proteins constitute one of the largest protein classes, with highly varied function. Consequently, the biosynthesis of Fe/S clusters is evolutionarily conserved and mutations in intermediate Fe/S cluster scaffold proteins can cause disease, including multiple mitochondrial dysfunctions syndrome (MMDS). Herein, we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (p.Gly189Arg) near the active site of NFU1, an Fe/S scaffold protein. In vitro investigation into the structure-function relationship of the Gly189Arg deriv., along with two other variants, reveals that substitution at position 189 triggers structural changes that increase flexibility, decrease stability, and alter the monomer-dimer equil. toward monomer, thereby impairing the ability of the Gly189X derivs. to receive an Fe/S cluster from physiol. relevant sources.
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      Jumper, J. Highly accurate protein structure prediction with AlphaFold. Nature 2021, 596 (7873), 583– 589,  DOI: 10.1038/s41586-021-03819-2
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      Highly accurate protein structure prediction with AlphaFold
      Jumper, John; Evans, Richard; Pritzel, Alexander; Green, Tim; Figurnov, Michael; Ronneberger, Olaf; Tunyasuvunakool, Kathryn; Bates, Russ; Zidek, Augustin; Potapenko, Anna; Bridgland, Alex; Meyer, Clemens; Kohl, Simon A. A.; Ballard, Andrew J.; Cowie, Andrew; Romera-Paredes, Bernardino; Nikolov, Stanislav; Jain, Rishub; Adler, Jonas; Back, Trevor; Petersen, Stig; Reiman, David; Clancy, Ellen; Zielinski, Michal; Steinegger, Martin; Pacholska, Michalina; Berghammer, Tamas; Bodenstein, Sebastian; Silver, David; Vinyals, Oriol; Senior, Andrew W.; Kavukcuoglu, Koray; Kohli, Pushmeet; Hassabis, Demis
      Nature (London, United Kingdom) (2021), 596 (7873), 583-589CODEN: NATUAS; ISSN:0028-0836. (Nature Portfolio)
      Proteins are essential to life, and understanding their structure can facilitate a mechanistic understanding of their function. Through an enormous exptl. effort, the structures of around 100,000 unique proteins have been detd., but this represents a small fraction of the billions of known protein sequences. Structural coverage is bottlenecked by the months to years of painstaking effort required to det. a single protein structure. Accurate computational approaches are needed to address this gap and to enable large-scale structural bioinformatics. Predicting the three-dimensional structure that a protein will adopt based solely on its amino acid sequence-the structure prediction component of the 'protein folding problem'-has been an important open research problem for more than 50 years. Despite recent progress, existing methods fall far short of at. accuracy, esp. when no homologous structure is available. Here we provide the first computational method that can regularly predict protein structures with at. accuracy even in cases in which no similar structure is known. We validated an entirely redesigned version of our neural network-based model, AlphaFold, in the challenging 14th Crit. Assessment of protein Structure Prediction (CASP14), demonstrating accuracy competitive with exptl. structures in a majority of cases and greatly outperforming other methods. Underpinning the latest version of AlphaFold is a novel machine learning approach that incorporates phys. and biol. knowledge about protein structure, leveraging multi-sequence alignments, into the design of the deep learning algorithm.
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    82. 82
      Cai, K.; Frederick, R. O.; Markley, J. L. ISCU interacts with NFU1, and ISCU[4Fe-4S] transfers its Fe-S cluster to NFU1 leading to the production of holo-NFU1. J. Struct Biol. 2020, 210 (2), 107491,  DOI: 10.1016/j.jsb.2020.107491
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      82
      ISCU interacts with NFU1, and ISCU[4Fe-4S] transfers its Fe-S cluster to NFU1 leading to the production of holo-NFU1
      Cai, Kai; Frederick, Ronnie O.; Markley, John L.
      Journal of Structural Biology (2020), 210 (2), 107491CODEN: JSBIEM; ISSN:1047-8477. (Elsevier Inc.)
      NFU1 is a late-acting factor in the biogenesis of human mitochondrial iron-sulfur proteins. Mutations in NFU1 are assocd. with genetic diseases such as multiple mitochondrial dysfunctions syndrome 1 (MMDS1) that involve defects in mitochondrial [4Fe-4S] proteins. We present results from NMR spectroscopy, small angle X-ray scattering, size exclusion chromatog., and isothermal titrn. calorimetry showing that the structured conformer of human ISCU binds human NFU1. The dissocn. const. detd. by ITC is Kd = 1.1 ± 0.2 μM. NMR and SAXS studies led to a structural model for the complex in which the cluster binding region of ISCU interacts with two α-helixes in the C-terminal domain of NFU1. In vitro expts. demonstrate that ISCU[4Fe-4S] transfers its Fe-S cluster to apo-NFU1, in the absence of a chaperone, leading to the assembly of holo-NFU1. By contrast, the cluster of ISCU[2Fe-2S] remains bound to ISCU in the presence of apo-NFU1.
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    83. 83
      Uzarska, M. A; Nasta, V.; Weiler, B. D; Spantgar, F.; Ciofi-Baffoni, S.; Saviello, M. R.; Gonnelli, L.; Muhlenhoff, U.; Banci, L.; Lill, R. Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins. Elife 2016, 5, e16673,  DOI: 10.7554/eLife.16673
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      84
      Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins
      Uzarska, Marta A.; Nasta, Veronica; Weiler, Benjamin D.; Spantgar, Farah; CiofiBaffoni, Simone; Saviello, Maria Rosaria; Gonnelli, Leonardo; Muhlenhoff, Ulrich; Banci, Lucia; Lill, Roland
      eLife (2016), 5 (), e16673/1-e16673/25, S1-S8CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochem. basis for explaining the pathol. phenotypes of patients with mutations in BOLA3.
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    84. 84
      Sheftel, A. D. The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation. Mol. Biol. Cell 2012, 23 (7), 1157– 66,  DOI: 10.1091/mbc.e11-09-0772
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      The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation
      Sheftel, Alex D.; Wilbrecht, Claudia; Stehling, Oliver; Niggemeyer, Brigitte; Elsaesser, Hans-Peter; Muehlenhoff, Ulrich; Lill, Roland
      Molecular Biology of the Cell (2012), 23 (7), 1157-1166CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
      Members of the bacterial and mitochondrial iron-sulfur cluster (ISC) assembly machinery include the so-called A-type ISC proteins, which support the assembly of a subset of Fe/S apoproteins. The human genome encodes two A-type proteins, termed ISCA1 and ISCA2, which are related to Saccharomyces cerevisiae Isa1 and Isa2, resp. An addnl. protein, Iba57, phys. interacts with Isa1 and Isa2 in yeast. To test the cellular role of human ISCA1, ISCA2, and IBA57, HeLa cells were depleted for any of these proteins by RNA interference technol. Depleted cells contained massively swollen and enlarged mitochondria that were virtually devoid of cristae membranes, demonstrating the importance of these proteins for mitochondrial biogenesis. The activities of mitochondrial [4Fe-4S] proteins, including aconitase, respiratory complex I, and lipoic acid synthase, were diminished following depletion of the three proteins. In contrast, the mitochondrial [2Fe-2S] enzyme ferrochelatase and cellular heme content were unaffected. We further provide evidence against a localization and direct Fe/S protein maturation function of ISCA1 and ISCA2 in the cytosol. Taken together, our data suggest that ISCA1, ISCA2, and IBA57 are specifically involved in the maturation of mitochondrial [4Fe-4S] proteins functioning late in the ISC assembly pathway.
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    85. 85
      Beinert, H.; Holm, R. H.; Münck, E. Iron-sulfur clusters: nature’s modular, multipurpose structures. Science 1997, 277, 653– 659,  DOI: 10.1126/science.277.5326.653
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      Iron-sulfur clusters: nature's modular, multipurpose structures
      Beinert, Helmut; Holm, Richard H.; Munck, Eckard
      Science (Washington, D. C.) (1997), 277 (5326), 653-659CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      A review with 78 refs. Iron-sulfur proteins are found in all life forms. Most frequently, they contain Fe2S2, Fe3S4, and Fe4S4 clusters. These modular clusters undergo oxidn.-redn. reactions, may be inserted or removed from proteins, can influence protein structure by preferential side chain ligation, and can be interconverted. In addn. to their electron transfer function, iron-sulfur clusters act as catalytic centers and sensors of iron and oxygen. Their most common oxidn. states are paramagnetic and present significant challenges for understanding the magnetic properties of mixed valence systems. Iron-sulfur clusters now rank with such biol. prosthetic groups as hemes and flavins in pervasive occurrence and multiplicity function.
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    86. 86
      Johnson, M. K. Iron–Sulfur Proteins: New Roles for Old Clusters. Curr. Opin. Chem. Biol. 1998, 2, 173– 181,  DOI: 10.1016/S1367-5931(98)80058-6
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      Iron-sulfur proteins: new roles for old clusters
      Johnson, Michael K.
      Current Opinion in Chemical Biology (1998), 2 (2), 173-181CODEN: COCBF4; ISSN:1367-5931. (Current Biology Ltd.)
      A review with 64 refs. Several major advances in our understanding of the structure, function and properties of biol. iron-sulfur clusters have occurred in the past year. These include a new structural type of cluster in the inappropriately named prismane protein, the establishment of redox-mediated [Fe2S2]2+ ↔ [Fe4S4]2+ cluster conversions, and the characterization of valence-delocalized [Fe2S2]+ and all ferrous clusters with [Fe2S2]0, [Fe3S4]2- and [Fe4S4]0 cores. The emergence of novel types of redox, regulatory and enzymic roles have also raised the possibility of iron-sulfur clusters mediating two electron redox processes, coupling proton and electron transfer, and catalyzing disulfide redn. and reductive cleavage of S-adenosylmethionine via sulfur-based cluster chem.
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    87. 87
      Honarmand Ebrahimi, K. Iron-sulfur clusters as inhibitors and catalysts of viral replication. Nat. Chem. 2022, 14 (3), 253– 266,  DOI: 10.1038/s41557-021-00882-0
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      88
      Iron-sulfur clusters as inhibitors and catalysts of viral replication
      Honarmand Ebrahimi, Kourosh; Ciofi-Baffoni, Simone; Hagedoorn, Peter-Leon; Nicolet, Yvain; Le Brun, Nick E.; Hagen, Wilfred R.; Armstrong, Fraser A.
      Nature Chemistry (2022), 14 (3), 253-266CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)
      A review. A virus hijacks host cellular machineries and metabolites in order to reproduce. In response, the innate immune system activates different processes to fight back. Although many aspects of these processes have been well investigated, the key roles played by iron-sulfur [FeS] clusters, which are among the oldest classes of bio-inorg. cofactors, have barely been considered. Here we discuss how several [FeS] cluster-contg. proteins activate, support and modulate the innate immune response to restrict viral infections, and how some of these proteins simultaneously support the replication of viruses. We also propose models of function of some proteins in the innate immune response and argue that [FeS] clusters in many of these proteins act as biol. 'fuses' to control the response. We hope this overview helps to inspire future research in the emerging field of bio-inorg. virol./immunol. and that such studies may reveal new mol. insight into the links between viral infections and diseases like cancer and neurodegeneration.
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      Pritts, J. D.; Michel, S. L. J. Fe-S clusters masquerading as zinc finger proteins. J. Inorg. Biochem 2022, 230, 111756,  DOI: 10.1016/j.jinorgbio.2022.111756
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      Fe-S clusters masquerading as zinc finger proteins
      Pritts, Jordan D.; Michel, Sarah L. J.
      Journal of Inorganic Biochemistry (2022), 230 (), 111756CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Inc.)
      A review. Metal ions are commonly found as protein co-factors in biol., and it is estd. that over a quarter of all proteins require a metal cofactor. The distribution and utilization of metals in biol. has changed over time. As the earth evolved, the atm. became increasingly oxygen rich which affected the bioavailability of certain metals such as iron, which in the oxidized ferric form is significantly less sol. than its reduced ferrous counterpart. Addnl., proteins that utilize metal cofactors for structural purposes grew in abundance, necessitating the use of metal co-factors that are not redox active, such as zinc. One common class of Zn co-factored proteins are zinc finger proteins (ZFs). ZFs bind zinc utilizing cysteine and histidine ligands to promote structure and function. Bioinformatics has annotated 5% of the human genome as ZFs; however, many of these proteins have not been studied empirically. In recent years, examples of annotated ZFs that instead harbor Fe-S clusters have been reported. In this review we highlight four examples of mis-annotated ZFs: mitoNEET, CPSF30, nsp12, and Fep1 and describe methods that can be utilized to differentiate the metal-cofactor.
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    89. 89
      Cameron, J. M.; Janer, A.; Levandovskiy, V.; MacKay, N.; Rouault, T. A.; Tong, W.-H.; Ogilvie, I.; Shoubridge, E. A.; Robinson, B. H. Mutations in iron-sulfur cluster scaffold genes NFU1 and BOLA3 cause a fatal deficiency of multiple respiratory chain and 2-oxoacid dehydrogenase enzymes. Am. J. Hum. Genet. 2011, 89, 486– 495,  DOI: 10.1016/j.ajhg.2011.08.011
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      90
      Mutations in Iron-Sulfur Cluster Scaffold Genes NFU1 and BOLA3 Cause a Fatal Deficiency of Multiple Respiratory Chain and 2-Oxoacid Dehydrogenase Enzymes
      Cameron, Jessie M.; Janer, Alexandre; Levandovskiy, Valeriy; MacKay, Nevena; Rouault, Tracey A.; Tong, Wing-Hang; Ogilvie, Isla; Shoubridge, Eric A.; Robinson, Brian H.
      American Journal of Human Genetics (2011), 89 (4), 486-495CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
      Severe combined deficiency of the 2-oxoacid dehydrogenases, assocd. with a defect in lipoate synthesis and accompanied by defects in complexes I, II, and III of the mitochondrial respiratory chain, is a rare autosomal recessive syndrome with no obvious causative gene defect. A candidate locus for this syndrome was mapped to chromosomal region 2p14 by microcell-mediated chromosome transfer in two unrelated families. Unexpectedly, anal. of genes in this area identified mutations in two different genes, both of which are involved in [Fe-S] cluster biogenesis. A homozygous missense mutation, c.545G>A, near the splice donor of exon 6 in NFU1 predicting a p.Arg182Gln substitution was found in one of the families. The mutation results in abnormal mRNA splicing of exon 6, and no mature protein could be detected in fibroblast mitochondria. A single base-pair duplication c.123dupA was identified in BOLA3 in the second family, causing a frame shift that produces a premature stop codon (p.Glu42Argfs*13). Transduction of fibroblast lines with retroviral vectors expressing the mitochondrial, but not the cytosolic isoform of NFU1 and with isoform 1, but not isoform 2 of BOLA3 restored both respiratory chain function and oxoacid dehydrogenase complexes. NFU1 was previously proposed to be an alternative scaffold to ISCU for the biogenesis of [Fe-S] centers in mitochondria, and the function of BOLA3 was previously unknown. Our results demonstrate that both play essential roles in the prodn. of [Fe-S] centers for the normal maturation of lipoate-contg. 2-oxoacid dehydrogenases, and for the assembly of the respiratory chain complexes.
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      Lossos, A. Fe/S protein assembly gene IBA57 mutation causes hereditary spastic paraplegia. Neurology 2015, 84 (7), 659– 67,  DOI: 10.1212/WNL.0000000000001270
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      Fe/S protein assembly gene IBA57 mutation causes hereditary spastic paraplegia
      Lossos, Alexander; Stuempfig, Claudia; Stevanin, Giovanni; Gaussen, Marion; Zimmerman, Bat-El; Mundwiller, Emeline; Asulin, Moriya; Chamma, Liat; Sheffer, Ruth; Misk, Adel; Dotan, Shlomo; Gomori, John M.; Ponger, Penina; Brice, Alexis; Lerer, Israela; Meiner, Vardiella; Lill, Roland
      Neurology (2015), 84 (7), 659-667CODEN: NEURAI; ISSN:0028-3878. (Lippincott Williams & Wilkins)
      Objective: To present the clin., mol., and cell biol. findings in a family with an autosomal recessive form of hereditary spastic paraplegia characterized by a combination of spastic paraplegia, optic atrophy, and peripheral neuropathy (SPOAN). Methods: We used a combination of whole-genome linkage anal. and exome sequencing to map the disease locus and to identify the responsible gene. To analyze the physiol. consequences of the disease, we used biochem. and cell biol. methods. Results: Ten members of a highly consanguineous family manifested a childhood-onset SPOAN-like phenotype with slow progression into late adulthood. We mapped this disorder to a locus on chromosome 1q and identified a homozygous donor splice-site mutation in the IBA57 gene, previously implicated in 2 infants with lethal perinatal encephalomyopathy. This gene encodes the mitochondrial iron-sulfur (Fe/S) protein assembly factor IBA57. In addn. to a severely decreased amt. of normal IBA57 mRNA, a patient's cells expressed an aberrantly spliced mRNA with a premature stop codon. Lymphoblasts contained 10-fold-lower levels of wild-type, but no signs of truncated IBA57 protein. The decrease in functional IBA57 resulted in reduced levels and activities of several mitochondrial [4Fe-4S] proteins, including complexes I and II, while mitochondrial [2Fe-2S] proteins remained normal. Conclusions: Our findings reinforce the suggested specific function of IBA57 in mitochondrial [4Fe-4S] protein maturation and provide addnl. evidence for its role in human disease. The less decreased IBA57 protein level in this family explains phenotypic differences compared with the previously described lethal encephalomyopathy with no functional IBA57.
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      Debray, F. G. Mutation of the iron-sulfur cluster assembly gene IBA57 causes fatal infantile leukodystrophy. J. Inherit Metab Dis 2015, 38 (6), 1147– 53,  DOI: 10.1007/s10545-015-9857-1
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      Mutation of the iron-sulfur cluster assembly gene IBA57 causes fatal infantile leukodystrophy
      Debray, Francois-Guillaume; Stumpfig, Claudia; Vanlander, Arnaud V.; Dideberg, Vinciane; Josse, Claire; Caberg, Jean-Hubert; Boemer, Francois; Bours, Vincent; Stevens, Rene; Seneca, Sara; Smet, Joel; Lill, Roland; van Coster, Rudy
      Journal of Inherited Metabolic Disease (2015), 38 (6), 1147-1153CODEN: JIMDDP; ISSN:0141-8955. (Springer)
      Leukodystrophies are a heterogeneous group of severe genetic neurodegenerative disorders. A multiple mitochondrial dysfunctions syndrome was found in an infant presenting with a progressive leukoencephalopathy. Homozygosity mapping, whole exome sequencing, and functional studies were used to define the underlying mol. defect. Respiratory chain studies in skeletal muscle isolated from the proband revealed a combined deficiency of complexes I and II. In addn., western blotting indicated lack of protein lipoylation. The combination of these findings was suggestive for a defect in the iron-sulfur (Fe/S) protein assembly pathway. SNP array identified loss of heterozygosity in large chromosomal regions, covering the NFU1 and BOLA3, and the IBA57 and ABCB10 candidate genes, in 2p15-p11.2 and 1q31.1-q42.13, resp. A homozygous c.436C > T (p.Arg146Trp) variant was detected in IBA57 using whole exome sequencing. Complementation studies in a HeLa cell line depleted for IBA57 showed that the mutant protein with the semi-conservative amino acid exchange was unable to restore the biochem. phenotype indicating a loss-of-function mutation of IBA57. In conclusion, defects in the Fe/S protein assembly gene IBA57 can cause autosomal recessive neurodegeneration assocd. with progressive leukodystrophy and fatal outcome at young age. In the affected patient, the biochem. phenotype was characterized by a defect in the respiratory chain complexes I and II and a decrease in mitochondrial protein lipoylation, both resulting from impaired assembly of Fe/S clusters.
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    92. 92
      Iwig, D. F.; Booker, S. J. Insight into the polar reactivity of the onium chalcogen analogues of S-adenosyl-L-methionine. Biochemistry 2004, 43 (42), 13496– 13509,  DOI: 10.1021/bi048693+
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      Insight into the Polar Reactivity of the Onium Chalcogen Analogues of S-Adenosyl-L-methionine
      Iwig, David F.; Booker, Squire J.
      Biochemistry (2004), 43 (42), 13496-13509CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      S-Adenosyl-L-methionine (AdoMet) is one of Nature's most diverse metabolites, used not only in a large no. of biol. reactions but amenable to several different modes of reactivity. The types of transformations in which it is involved include decarboxylation, electrophilic addn. to any of the three carbons bonded to the central sulfur atom, proton removal at carbons adjacent to the sulfonium, and reductive cleavage to generate 5'-deoxyadenosyl 5'-radical intermediates. At physiol. pH and temp., AdoMet is subject to three spontaneous degrdn. pathways, the first of which is racemization of the chiral sulfonium group, which takes place in a pH-independent manner. The two remaining pathways are pH-dependent and include (1) intramol. attack of the α-carboxylate group onto the γ-carbon, affording L-homoserine lactone (HSL) and 5'-methylthioadenosine (MTA), and (2) deprotonation at C-5', initiating a cascade that results in formation of adenine and S-ribosylmethionine. Herein, we describe pH-dependent stability studies of AdoMet and its selenium and tellurium analogs, Se-adenosyl-L-selenomethionine and Te-adenosyl-L-telluromethionine (SeAdoMet and TeAdoMet, resp.), at 37° and const. ionic strength, which we use as a probe of their relative intrinsic reactivities. We find that with AdoMet intramol. nucleophilic attack to afford HSL and MTA exhibits a pH-rate profile having two titratable groups with apparent pKa values of 1.2±0.4 and 8.2±0.05 and displaying first-order rate consts. of <0.7×10-6 s-1 at pH values less than 0.5, ∼3×10-6 s-1 at pH values between 2 and 7, and ∼15×10-6 s-1 at pH values greater than 9. Degrdn. via deprotonation at C-5' follows a pH-rate profile having one titratable group with an apparent pKa value of ∼11.5. The selenium analog decays significantly faster via intramol. nucleophilic attack, also exhibiting a pH-rate profile with two titratable groups with pKa values of ∼0.86 and 8.0±0.1 with first-order rate consts. of <7×10-6 s-1 at pH values less than 0.9, ∼32×10-6 s-1 at pH values between 2 and 7, and ∼170×10-6 s-1 at pH values greater than 9. Degrdn. via deprotonation at C-5' proceeds with one titratable group displaying an apparent pKa value of ∼14.1. Unexpectedly, TeAdoMet did not decay at an observable rate via either of these two pathways. Last, enzymically synthesized AdoMet was found to racemize at rates that were consistent with earlier studies (Hoffman, J. L. (1986) Biochem. 25, 4444-4449); however, SeAdoMet and TeAdoMet did not racemize at detectable rates. In the accompanying paper, we use the information obtained in these model studies to probe the mechanism of cyclopropane fatty acid synthase via use of the onium chalcogens of AdoMet as Me donors.
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      Lanz, N. D. RlmN and AtsB as models for the overproduction and characterization of radical SAM proteins. Methods Enzymol. 2012, 516, 125– 152,  DOI: 10.1016/B978-0-12-394291-3.00030-7
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      RlmN and AtsB as models for the overproduction and characterization of radical SAM proteins
      Lanz, Nicholas D.; Grove, Tyler L.; Gogonea, Camelia Baleanu; Lee, Kyung-Hoon; Krebs, Carsten; Booker, Squire J.
      Methods in Enzymology (2012), 516 (Natural Product Biosynthesis by Microorganisms and Plants, Part B), 125-152CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)
      A review. An explosion of remarkable chem. transformations has been witnessed in the past decade as a result of the radical S-adenosyl-L-methionine (SAM) (RS) superfamily of proteins. These proteins share the ability to cleave SAM reductively to L-methionine and a 5'-deoxyadenosyl 5'-radical (5'-dA*). The 5'-dA* initiates >40 distinct reaction types by abstracting target hydrogen atoms on small-mol. and macromol. substrates. All RS enzymes contain a [4Fe-4S] cluster coordinated by SAM that supplies the electron for SAM cleavage. A subset of RS enzymes contains addnl. iron-sulfur (Fe/S) clusters that serve alternative purposes, many remaining to be defined. The oxygen lability of their [4Fe-4S] clusters causes RS enzymes to be more tedious to purify, characterize, and study. Moreover, the type(s) and stoichiometry of Fe/S clusters in RS enzymes has often been a source of debate. Herein, we use RlmN and AtsB as models to highlight methods for purifying and characterizing RS enzymes, focusing on using Mossbauer spectroscopy in concert with methods for quantifying iron and acid-labile sulfide to assign cluster content accurately.
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      Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 1976, 72, 248– 254,  DOI: 10.1016/0003-2697(76)90527-3
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      A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
      Bradford, Marion M.
      Analytical Biochemistry (1976), 72 (1-2), 248-54CODEN: ANBCA2; ISSN:0003-2697.
      A protein detn. method that involves the binding of coomassie Brilliant Blue G 250 to protein is described. The binding of the dye to protein causes a shift in the absorption max. of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm that is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in ∼ 2 min with good color stability for 1 hr. There is little or no interference from cations such as Na+ or K+ nor from carbohydrates such as sucrose. A small amt. of color is developed in the presence of strongly alk. buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amts. of detergents such as Na dodecyl sulfate, Triton X 100, and commercial glassware detergents. Interference by small amts. of detergent may be eliminated by the use of proper control.
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      Beinert, H. Micro methods for the quantitative determination of iron and copper in biological material. Methods Enzymol. 1978, 54, 435– 445,  DOI: 10.1016/S0076-6879(78)54027-5
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      Micro methods for the quantitative determination of iron and copper in biological material
      Beinert, Helmut
      Methods in Enzymology (1978), 54 (), 435-45CODEN: MENZAU; ISSN:0076-6879.
      A micro method for detn. of Fe and Cu in biol. materials is described that is aimed at those who have no established method in use and who prefer chem. detns. rather than purely spectroscopic ones. Essential features of the method are wet ashing, evapn. of excess acid, redn., neutralization with excess Na acetate, development of color with a suitable bathophenanthroline, and extn. with a small quantity of org. solvent followed by spectrometric detn.
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      Beinert, H. Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. Anal. Biochem. 1983, 131, 373– 378,  DOI: 10.1016/0003-2697(83)90186-0
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      Semimicro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins
      Beinert, Helmut
      Analytical Biochemistry (1983), 131 (2), 373-8CODEN: ANBCA2; ISSN:0003-2697.
      The detn. of labile sulfide in Fe-S proteins in the range of 1 to 3 nmol is described. Analyses are also presented on the most stable Fe-S protein so far reported. In this case denaturation with guanidine-HCl was used in the presence of dithiothreitol. The values obtained then also include any sulfane S (S0) present. The colorimetric procedure is modified from that of J. K. Fogo and M. Popowsky (1949).
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      Kennedy, M. C. Evidence for the Formation of a Linear [3Fe-4S] Cluster in Partially Unfolded Aconitase. J. Biol. Chem. 1984, 259 (23), 14463– 14471,  DOI: 10.1016/S0021-9258(17)42622-6
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      Evidence for the formation of a linear [iron-sulfur] ([3Fe-4S]) cluster in partially unfolded aconitase
      Kennedy, Mary Claire; Kent, Thomas A.; Emptage, Mark; Merkle, Hellmut; Beinert, Helmut; Munck, Eckard
      Journal of Biological Chemistry (1984), 259 (23), 14463-71CODEN: JBCHA3; ISSN:0021-9258.
      Beef heart aconitase, as isolated under aerobic conditions, is inactive and contains a [3Fe-4S]+ cluster. On incubation at pH >9.5 (or treatment with 4-8M urea), the color of the protein changes from brown to purple. This purple form is stable and can be converted back in good yield to the active [4Fe-4S]2+ form by redn. in the presence of Fe. Active aconitase is converted to the purple form at alk. pH only after oxidative inactivation. The Fe/S2- ratio of purple aconitase is 0.8, indicating the presence of [3Fe-4S] clusters. The no. of SH groups readily reacting with 5,5'-dithiobis(2-nitrobenzoic acid) is increased from ∼1 in the enzyme as isolated to 7-8 in the purple form, indicating a partial unfolding of the protein. On conversion of inactive aconitase to the purple form, the ESR signal at g = 2.01 (S (spin) = 1/2) is replaced by signals at g = 4.3 and 9.6 (S = 5/2). Moessbauer spectroscopy shows that purple aconitase has high-spin Fe3+ ions, each residing in a tetrahedral environment of S atoms. The 3 Fe sites are exchange-coupled to yield a ground state with S = 5/2. Anal. of the data with spin coupling shows that the spin-coupling consts. J13 ≃ J23 and 2 J12 < J13, where the Jik describe the antiferromagnetic (J >0) exchange interactions among the 3 Fe pairs. Comparison of these data with those reported for synthetic Fe-S clusters (Hagen, K. S., et al., 1983) shows that purple aconitase contains a linear [3Fe-4S]+ cluster, a structural isomer of the S = 1/2 cluster of inactive aconitase. Also, protein-bound [2Fe-2S] clusters evidently can be generated under conditions where partial unfolding of the protein occurs.
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      Blaszczyk, A. J. Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase. J. Am. Chem. Soc. 2016, 138 (10), 3416– 3426,  DOI: 10.1021/jacs.5b12592
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      Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase
      Blaszczyk, Anthony J.; Silakov, Alexey; Zhang, Bo; Maiocco, Stephanie J.; Lanz, Nicholas D.; Kelly, Wendy L.; Elliott, Sean J.; Krebs, Carsten; Booker, Squire J.
      Journal of the American Chemical Society (2016), 138 (10), 3416-3426CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      TsrM, an annotated radical S-adenosylmethionine (SAM) enzyme, catalyzes the methylation of carbon 2 of the indole ring of L-tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended Me group derives from SAM; however, the enzyme also requires cobalamin and iron-sulfur cluster cofactors for turnover. In this work we report the overprodn. and purifn. of TsrM and the characterization of its metallocofactors by UV-visible, ESR, hyperfine sublevel correlation (HYSCORE), and M.ovrddot.ossbauer spectroscopies as well as protein-film electrochem. (PFE). The enzyme contains 1 equiv of its cobalamin cofactor in its as-isolated state and can be reconstituted with iron and sulfide to contain one [4Fe-4S] cluster with a site-differentiated Fe2+/Fe3+ pair. Our spectroscopic studies suggest that TsrM binds cobalamin in an uncharacteristic five-coordinate base-off/His-off conformation, whereby the dimethylbenzimidazole group is replaced by a non-nitrogenous ligand, which is likely a water mol. Electrochem. anal. of the protein by PFE indicates a one-electron redox feature with a midpoint potential of -550 mV, which is assigned to a [4Fe-4S]2+/[4Fe-4S]+ redox couple. Anal. of TsrM by M.ovrddot.ossbauer and HYSCORE spectroscopies suggests that SAM does not bind to the unique iron site of the cluster in the same manner as in other radical SAM (RS) enzymes, yet its binding still perturbs the electronic configuration of both the Fe/S cluster and the cob(II)alamin cofactors. These biophys. studies suggest that TsrM is an atypical RS enzyme, consistent with its reported inability to catalyze formation of a 5'-deoxyadenosyl 5'-radical.
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