Metabolic Pathway Rerouting in Paraburkholderia rhizoxinica Evolved Long-Overlooked Derivatives of Coenzyme F₄₂₀

Click to copy article linkArticle link copied!

This article references 37 other publications.

1. 1

   Jacobson, F. and Walsh, C. (1984) Properties of 7,8-didemethyl-8-hydroxy-5-deazaflavins relevant to redox coenzyme function in methanogen metabolism. Biochemistry 23, 979– 988,  DOI: 10.1021/bi00300a028

   Google Scholar

   1

   Properties of 7,8-didemethyl-8-hydroxy-5-deazaflavins relevant to redox coenzyme function in methanogen metabolism

   Jacobson, Fredric; Walsh, Christopher

   Biochemistry (1984), 23 (5), 979-88CODEN: BICHAW; ISSN:0006-2960.

   The 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) moiety is the key element in the redox coenzyme factor 420 (F420) found in methanogenic bacteria and in streptomycetes. In this paper, the chem. properties of synthetic FO that condition coenzyme function are analyzed, and FO is compared with 5-deazariboflavin (5-dRF) and 8-hydroxyriboflavin. The equil. consts. for sulfite addn. and the rates of reoxidn. of FOH2 by a series of flavin analogs show that the 5-carba substitution imposes nicotinamide-like chem. on the system, including sluggishness to reoxidn. by O2. Ionization of the 8-OH substituent in the oxidized FO (pKa = 5.85) suppresses reactivity of FO toward redox chem. In the reduced form, FOH2, the phenolic group is isolated and shows a more normal pKa of 9.7. The redn. potential of FO/FOH2 was detd. by equilibration with 2 methanogen enzymes, an F420-reducing hydrogenase and an F420-NADP reductase, to be -340 to -350 mV. The rate of the bimol. disproportionation of FOH2 and FO was followed by high-pressure liq. chromatog. anal., starting with 3H in the oxidized species, and shown to be 10-20 M-1 min-1, down 50-100-fold from the 5-dRF/5-dRFH2 reaction. This extended lifetime of chiral [5-3H]FOH2 samples in the presence of FO mols. permits stereochem. detn. of hydride transfers to and from C(5) of the 8-hydroxy-5-deazaflavin system. Methanogen hydrogenase and F420-NADP reductase are defined to show A side specificity, whereas the NADPH-dependent FMN reductase from Beneckea harveyi shows B side specificity.
2. 2

   Greening, C., Ahmed, F. H., Mohamed, A. E., Lee, B. M., Pandey, G., Warden, A. C., Scott, C., Oakeshott, J. G., Taylor, M. C., and Jackson, C. J. (2016) Physiology, biochemistry, and applications of F₄₂₀- and F_o-dependent redox reactions. Microbiol. Mol. Biol. Rev. 80, 451– 493,  DOI: 10.1128/MMBR.00070-15

   Google Scholar

   2

   Physiology, biochemistry, and applications of F420- and Fo-dependent redox reactions

   Greening, Chris; Ahmed, F. Hafna; Mohamed, A. Elaaf; Lee, Brendon M.; Pandey, Gunjan; Warden, Andrew C.; Scott, Colin; Oakeshott, John G.; Taylor, Matthew C.; Jackson, Colin J.

   Microbiology and Molecular Biology Reviews (2016), 80 (2), 451-493CODEN: MMBRF7; ISSN:1098-5557. (American Society for Microbiology)

   5-Deazaflavin cofactors enhance the metabolic flexibility of microorganisms by catalyzing a wide range of challenging enzymic redox reactions. While structurally similar to riboflavin, 5-deazaflavins have distinctive and biol. useful electrochem. and photochem. properties as a result of the substitution of N-5 of the isoalloxazine ring for a carbon. 8-Hydroxy-5-deazaflavin (Fo) appears to be used for a single function: as a light-harvesting chromophore for DNA photolyases across the three domains of life. In contrast, its oligoglutamyl deriv. F420 is a taxonomically restricted but functionally versatile cofactor that facilitates many low-potential two-electron redox reactions. It serves as an essential catabolic cofactor in methanogenic, sulfate-reducing, and likely methanotrophic archaea. It also transforms a wide range of exogenous substrates and endogenous metabolites in aerobic actinobacteria, for example mycobacteria and streptomycetes. In this review, we discuss the physiol. roles of F420 in microorganisms and the biochem. of the various oxidoreductases that mediate these roles. Particular focus is placed on the central roles of F420 in methanogenic archaea in processes such as substrate oxidn., C1 pathways, respiration, and oxygen detoxification. We also describe how two F420-dependent oxidoreductase superfamilies mediate many environmentally and medically important reactions in bacteria, including biosynthesis of tetracycline and pyrrolobenzodiazepine antibiotics by streptomycetes, activation of the prodrugs pretomanid and delamanid by Mycobacterium tuberculosis, and degrdn. of environmental contaminants such as picrate, aflatoxin, and malachite green. The biosynthesis pathways of Fo and F420 are also detailed. We conclude by considering opportunities to exploit deazaflavin-dependent processes in tuberculosis treatment, methane mitigation, bioremediation, and industrial biocatalysis.
3. 3

   Purwantini, E. and Mukhopadhyay, B. (2009) Conversion of NO₂ to NO by reduced coenzyme F420 protects mycobacteria from nitrosative damage. Proc. Natl. Acad. Sci. U. S. A. 106, 6333– 6338,  DOI: 10.1073/pnas.0812883106

   Google Scholar

   3

   Conversion of NO2 to NO by reduced coenzyme F420 protects mycobacteria from nitrosative damage

   Purwantini, Endang; Mukhopadhyay, Biswarup

   Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (15), 6333-6338CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

   In mycobacteria, F420, a deazaflavin deriv., acts as a hydride transfer coenzyme for an F420-specific glucose-6-phosphate dehydrogenase (Fgd). Physiol. relevant reactions in the mycobacteria that use Fgd-generated reduced F420 (F420H2) are unknown. In this work, F420H2 was found to be oxidized by NO only in the presence of oxygen. Further anal. demonstrated that NO2, produced from NO and O2, was the oxidant. UV-visible spectroscopic and NO-sensor-based analyses proved that F420H2 reduced NO2 to NO. This reaction could serve as a defense system for Mycobacterium tuberculosis, which is more sensitive to NO2 than NO under aerobic conditions. Activated macrophages produce NO, which in acidified phagosomes is converted to NO2. Hence, by converting NO2 back to NO with F420H2, M. tuberculosis could decrease the effectiveness of antibacterial action of macrophages; such defense would correspond to active tuberculosis conditions where the bacterium grows aerobically. This hypothesis was consistent with the observation that a mutant strain of Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, which either did not produce or could not reduce F420, was ≈4-fold more sensitive to NO2 than the wild-type strain. The phenomenon is reminiscent of the anticancer activity of γ-tocopherol, which reduces NO2 to NO and protects human cells from NO2-induced carcinogenesis.
4. 4

   Singh, R., Manjunatha, U., Boshoff, H. I., Ha, Y. H., Niyomrattanakit, P., Ledwidge, R., Dowd, C. S., Lee, I. Y., Kim, P., Zhang, L., Kang, S., Keller, T. H., Jiricek, J., and Barry, C. E., 3rd (2008) PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular NO release. Science 322, 1392– 1395,  DOI: 10.1126/science.1164571

   Google Scholar

   4

   PA-824 Kills Nonreplicating Mycobacterium tuberculosis by Intracellular NO Release

   Singh, Ramandeep; Manjunatha, Ujjini; Boshoff, Helena I. M.; Ha, Young Hwan; Niyomrattanakit, Pornwaratt; Ledwidge, Richard; Dowd, Cynthia S.; Lee, Ill Young; Kim, Pilho; Zhang, Liang; Kang, Sunhee; Keller, Thomas H.; Jiricek, Jan; Barry, Clifton E., III

   Science (Washington, DC, United States) (2008), 322 (5906), 1392-1395CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)

   Bicyclic nitroimidazoles, including PA-824, are exciting candidates for the treatment of tuberculosis. These prodrugs require intracellular activation for their biol. function. We found that Rv3547 is a deazaflavin-dependent nitroreductase (Ddn) that converts PA-824 into three primary metabolites; the major one is the corresponding des-nitroimidazole (des-nitro). When derivs. of PA-824 were used, the amt. of des-nitro metabolite formed was highly correlated with anaerobic killing of Mycobacterium tuberculosis (Mtb). Des-nitro metabolite formation generated reactive nitrogen species, including nitric oxide (NO), which are the major effectors of the anaerobic activity of these compds. Furthermore, NO scavengers protected the bacilli from the lethal effects of the drug. Thus, these compds. may act as intracellular NO donors and could augment a killing mechanism intrinsic to the innate immune system.
5. 5

   Wang, P., Bashiri, G., Gao, X., Sawaya, M. R., and Tang, Y. (2013) Uncovering the enzymes that catalyze the final steps in oxytetracycline biosynthesis. J. Am. Chem. Soc. 135, 7138– 7141,  DOI: 10.1021/ja403516u

   Google Scholar

   5

   Uncovering the Enzymes that Catalyze the Final Steps in Oxytetracycline Biosynthesis

   Wang, Peng; Bashiri, Ghader; Gao, Xue; Sawaya, Michael R.; Tang, Yi

   Journal of the American Chemical Society (2013), 135 (19), 7138-7141CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

   Tetracyclines are a group of natural products sharing a linearly fused four-ring scaffold, which is essential for their broad-spectrum antibiotic activities. Formation of the key precursor anhydrotetracycline 3 during oxytetracycline 1 biosynthesis has been previously characterized. However, the enzymic steps that transform 3 into 1, including the addnl. hydroxylation at C5 and the final C5a-C11a redn., have remained elusive. Here we report two redox enzymes, OxyS and OxyR, are sufficient to convert 3 to 1. OxyS catalyzes two sequential hydroxylations at C6 and C5 positions of 3 with opposite stereochem., while OxyR catalyzes the C5a-C11a redn. using F420 as a cofactor to produce 1. The crystal structure of OxyS was obtained to provide insights into the tandem C6- and C5-hydroxylation steps. The substrate specificities of OxyS and OxyR were shown to influence the relative ratio of 1 and tetracycline 2.
6. 6

   Li, W., Khullar, A., Chou, S., Sacramo, A., and Gerratana, B. (2009) Biosynthesis of sibiromycin, a potent antitumor antibiotic. Appl. Environ. Microbiol. 75, 2869– 2878,  DOI: 10.1128/AEM.02326-08

   Google Scholar

   6

   Biosynthesis of sibiromycin, a potent antitumor antibiotic

   Li, Wei; Khullar, Ankush; Chou, Shen Chieh; Sacramo, Ashley; Gerratana, Barbara

   Applied and Environmental Microbiology (2009), 75 (9), 2869-2878CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)

   Pyrrolobenzodiazepines, a class of natural products produced by actinomycetes, are sequence selective DNA alkylating compds. with significant antitumor properties. Among the pyrrolo[1,4]benzodiazepines (PBDs) sibiromycin, one of two identified glycosylated PBDs, displays the highest affinity for DNA and the most potent antitumor properties. Despite the promising antitumor properties clin. trials of sibiromycin were precluded by the cardiotoxicity effect in animals attributed to the presence of the C-9 hydroxyl group. As a first step toward the development of sibiromycin analogs, we have cloned and localized the sibiromycin gene cluster to a 32.7-kb contiguous DNA region. Cluster boundaries tentatively assigned by comparative genomics were verified by gene replacement expts. The sibiromycin gene cluster consisting of 26 open reading frames reveals a "modular" strategy in which the synthesis of the anthranilic and dihydropyrrole moieties is completed before assembly by the nonribosomal peptide synthetase enzymes. In addn., the gene cluster identified includes open reading frames encoding enzymes involved in sibirosamine biosynthesis, as well as regulatory and resistance proteins. Gene replacement and chem. complementation studies are reported to support the proposed biosynthetic pathway.
7. 7

   Ichikawa, H., Bashiri, G., and Kelly, W. L. (2018) Biosynthesis of the thiopeptins and identification of an F₄₂₀H₂-dependent dehydropiperidine reductase. J. Am. Chem. Soc. 140, 10749– 10756,  DOI: 10.1021/jacs.8b04238

   Google Scholar

   7

   Biosynthesis of the Thiopeptins and Identification of an F420H2-Dependent Dehydropiperidine Reductase

   Ichikawa, Hiro; Bashiri, Ghader; Kelly, Wendy L.

   Journal of the American Chemical Society (2018), 140 (34), 10749-10756CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

   Thiopeptins are highly decorated thiopeptide antibiotics similar in structure to thiostrepton A and harbor two unusual features. All thiopeptins contain a thioamide, a rare moiety among natural products, and a subset of thiopeptins present with a piperidine in the core macrocycle rather than the more oxidated dehydropiperidine or pyridine rings typically obsd. in the thiopeptides. Here, we report the identification of the thiopeptin biosynthetic gene (tpn) cluster in Streptomyces tateyamensis and the gene product, TpnL, which shows sequence similarity to (deaza)flavin-dependent oxidoreductases. Heterologous expression of TpnL in the thiostrepton A producer, Streptomyces laurentii, led to the prodn. of a piperidine-contg. analog. Binding studies revealed TpnL preferentially binds the deazaflavin cofactor, coenzyme F420, and in vitro reconstitution of TpnL activity confirmed that this enzyme is an F420H2-dependent dehydropiperidine reductase. The identification of TpnL and its activity establishes the basis for the piperidine-contg. series a thiopeptides, one of the five main structural groups of this diverse family of antibiotics.
8. 8

   Heiss, G., Hofmann, K. W., Trachtmann, N., Walters, D. M., Rouviere, P., and Knackmuss, H. J. (2002) npd gene functions of Rhodococcus (opacus) erythropolis HL PM-1 in the initial steps of 2,4,6-trinitrophenol degradation. Microbiology 148, 799– 806,  DOI: 10.1099/00221287-148-3-799

   Google Scholar

   8

   npd gene functions of Rhodococcus (opacus) erythropolis HL PM-1 in the initial steps of 2,4,6-trinitrophenol degradation

   Heiss, Gesche; Hofmann, Klaus W.; Trachtmann, Natalie; Walters, Dana M.; Rouviere, Pierre; Knackmuss, Hans-Joachim

   Microbiology (Reading, United Kingdom) (2002), 148 (3), 799-806CODEN: MROBEO; ISSN:1350-0872. (Society for General Microbiology)

   Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol (picric acid) or 2,4-dinitrophenol (2,4-DNP) as sole nitrogen source. A gene cluster involved in picric acid degrdn. was recently identified. The functional assignment of three of its genes, npdC, npdG and npdI, and the tentative functional assignment of a fourth one, npdH, is reported. The genes were expressed in Escherichia coli as His-tag fusion proteins that were purified by Ni-affinity chromatog. The enzyme activity of each protein was detd. by spectrophotometry and HPLC analyses. NpdI, a hydride transferase, catalyzes a hydride transfer from reduced F420 to the arom. ring of picric acid, generating the hydride σ-complex (hydride Meisenheimer complex) of picric acid (H--PA). Similarly, NpdI also transformed 2,4-DNP to the hydride σ-complex of 2,4-DNP. A second hydride transferase, NpdC catalyzed a subsequent hydride transfer to H--PA, to produce a dihydride σ-complex of picric acid (2H--PA). All three reactions required the activity of NpdG, an NADPH-dependent F420 reductase, for shuttling the hydride ions from NADPH to F420. NpdH converted 2H--PA to a hitherto unknown product, X. The results show that npdC, npdG and npdI play a key role in the initial steps of picric acid degrdn., and that npdH may prove to be important in the later stages.
9. 9

   Taylor, M. C., Jackson, C. J., Tattersall, D. B., French, N., Peat, T. S., Newman, J., Briggs, L. J., Lapalikar, G. V., Campbell, P. M., Scott, C., Russell, R. J., and Oakeshott, J. G. (2010) Identification and characterization of two families of F₄₂₀ H₂-dependent reductases from Mycobacteria that catalyse aflatoxin degradation. Mol. Microbiol. 78, 561– 575,  DOI: 10.1111/j.1365-2958.2010.07356.x

   Google Scholar

   9

   Identification and characterization of two families of F420H2-dependent reductases from Mycobacteria that catalyse aflatoxin degradation

   Taylor, Matthew C.; Jackson, Colin J.; Tattersall, David B.; French, Nigel; Peat, Thomas S.; Newman, Janet; Briggs, Lyndall J.; Lapalikar, Gauri V.; Campbell, Peter M.; Scott, Colin; Russell, Robyn J.; Oakeshott, John G.

   Molecular Microbiology (2010), 78 (3), 561-575CODEN: MOMIEE; ISSN:0950-382X. (Wiley-Blackwell)

   Aflatoxins are polyarom. mycotoxins that contaminate a range of food crops as a result of fungal growth and contribute to serious health problems in the developing world because of their toxicity and mutagenicity. Although relatively resistant to biotic degrdn., aflatoxins can be metabolized by certain species of Actinomycetales. However, the enzymic basis for their breakdown has not been reported until now. We have identified nine Mycobacterium smegmatis enzymes that utilize the deazaflavin cofactor F420H2 to catalyze the redn. of the α,β-unsatd. ester moiety of aflatoxins, activating the mols. for spontaneous hydrolysis and detoxification. These enzymes belong to two previously uncharacterized F420H2 dependent reductase (FDR-A and -B) families that are distantly related to the FMN dependent pyridoxamine 5'-phosphate oxidases (PNPOxs). We have solved crystal structures of an enzyme from each FDR family and show that they, like the PNPOxs, adopt a split barrel protein fold, although the FDRs also possess an extended and highly charged F420H2 binding groove. A general role for these enzymes in xenobiotic metab. is discussed, including the observation that the nitro-reductase Rv3547 from Mycobacterium tuberculosis that is responsible for the activation of bicyclic nitroimidazole prodrugs belongs to the FDR-A family.
10. 10

    Taylor, M., Scott, C., and Grogan, G. (2013) F₄₂₀-dependent enzymes - potential for applications in biotechnology. Trends Biotechnol. 31, 63– 64,  DOI: 10.1016/j.tibtech.2012.09.003

    Google Scholar

    10

    F420-dependent enzymes - potential for applications in biotechnology

    Taylor, Matthew; Scott, Colin; Grogan, Gideon

    Trends in Biotechnology (2013), 31 (2), 63-64CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)

    A review. The unusual deazaflavin coenzyme F420 is being found in an increasingly diverse range of organisms, environments, and biochem. contexts. Developments in the authors' under-standing of its structure, biosynthesis, and the roles it plays in enzymol. are leading to an increased understanding of the enzymes that contain F420 , and their possible application in fields such as biocatalysis and bioremediation. Recent advances in the development of tools for the prodn. and utilization of F420 suggest an increase in research activity in this area in the future.
11. 11

    Mathew, S., Trajkovic, M., Kumar, H., Nguyen, Q. T., and Fraaije, M. W. (2018) Enantio- and regioselective ene-reductions using F₄₂₀H₂-dependent enzymes. Chem. Commun. 54, 11208– 11211,  DOI: 10.1039/C8CC04449J

    Google Scholar

    11

    Enantio- and regioselective ene-reductions using F420H2-dependent enzymes

    Mathew, Sam; Trajkovic, Milos; Kumar, Hemant; Nguyen, Quoc-Thai; Fraaije, Marco W.

    Chemical Communications (Cambridge, United Kingdom) (2018), 54 (79), 11208-11211CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)

    In the past decade it has become clear that many microbes harbor enzymes that employ an unusual flavin cofactor, the F420 deazaflavin cofactor. Herein we show that F420-dependent reductases (FDRs) can successfully perform enantio-, regio- and chemoselective ene-redns. For the first time, we have demonstrated that F420H2-driven reductases can be used as biocatalysts for the redn. of α,β-unsatd. ketones and aldehydes with good conversions (>99%) and excellent regioselectivities and enantiomeric excesses (>99% ee). Noteworthily, FDRs typically display an opposite enantioselectivity when compared to the well established FMN-dependent Old Yellow Enzymes (OYEs).
12. 12

    Decamps, L., Philmus, B., Benjdia, A., White, R., Begley, T. P., and Berteau, O. (2012) Biosynthesis of F₀, precursor of the F₄₂₀ cofactor, requires a unique two radical-SAM domain enzyme and tyrosine as substrate. J. Am. Chem. Soc. 134, 18173– 18176,  DOI: 10.1021/ja307762b

    Google Scholar

    12

    Biosynthesis of F0, Precursor of the F420 Cofactor, Requires a Unique Two Radical-SAM Domain Enzyme and Tyrosine as Substrate

    Decamps, Laure; Philmus, Benjamin; Benjdia, Alhosna; White, Robert; Begley, Tadhg P.; Berteau, Olivier

    Journal of the American Chemical Society (2012), 134 (44), 18173-18176CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

    Cofactors play key roles in metabolic pathways. Among them F420 has proved to be a very attractive target for the selective inhibition of archaea and actinobacteria. Its biosynthesis, in a unique manner, involves a key enzyme, F0-synthase. This enzyme is a large monomer in actinobacteria, while it is constituted of two subunits in archaea and cyanobacteria. The purifn. of both types of F0-synthase and their in vitro activities are reported here. This study allows one to establish that F0-synthase, from both types, uses 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine as substrates but not 4-hydroxylphenylpyruvate as previously suggested. Furthermore, the data support the fact that F0-synthase generates two 5'-deoxyadenosyl radicals for catalysis which is unprecedented in reaction catalyzed by radical SAM enzymes.
13. 13

    Grochowski, L. L., Xu, H., and White, R. H. (2008) Identification and characterization of the 2-phospho-L-lactate guanylyltransferase involved in coenzyme F₄₂₀ biosynthesis. Biochemistry 47, 3033– 3037,  DOI: 10.1021/bi702475t

    Google Scholar

    13

    Identification and characterization of the 2-phospho-L-lactate guanylyltransferase involved in coenzyme F420 biosynthesis

    Grochowski, Laura L.; Xu, Huimin; White, Robert H.

    Biochemistry (2008), 47 (9), 3033-3037CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

    Coenzyme F420 is a hydride carrier cofactor functioning in methanogenesis. One step in the biosynthesis of coenzyme F420 involves the coupling of 2-phospho-L-lactate (LP) to 7,8-didemethyl-8-hydroxy-5-deazaflavin, the F420 chromophore. This condensation requires an initial activation of 2-phospho-L-lactate through a pyrophosphate linkage to GMP. Bioinformatic anal. identified an uncharacterized archaeal protein in the Methanocaldococcus jannaschii genome, MJ0887, which could be involved in this transformation. The predicted MJ0887-derived protein had domain similarity with other known nucleotidyltransferases. The MJ0887 gene was cloned and overexpressed, and the purified protein was found to catalyze the formation of lactyl-2-diphospho-5'-guanosine from LP and GTP. Kinetic consts. were detd. for the MJ0887-derived protein with both LP and GTP substrates and were as follows: Vmax = 3 μmol min-1 mg-1; GTP Kmapp = 56 μM, and kcat/Kmapp = 2 × 104 M-1 s-1; and LP Kmapp = 36 μM, and kcat/Kmapp = 4 × 104 M-1 s-1. The MJ0887 gene product was designated CofC to indicate its involvement in the 3rd step of coenzyme F420 biosynthesis.
14. 14

    Graupner, M., Xu, H., and White, R. H. (2002) Characterization of the 2-phospho-L-lactate transferase enzyme involved in coenzyme F(420) biosynthesis in Methanococcus jannaschii. Biochemistry 41, 3754– 3761,  DOI: 10.1021/bi011937v

    Google Scholar

    14

    Characterization of the 2-Phospho-L-lactate Transferase Enzyme Involved in Coenzyme F420 Biosynthesis in Methanococcus jannaschii

    Graupner, Marion; Xu, Huimin; White, Robert H.

    Biochemistry (2002), 41 (11), 3754-3761CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

    The protein product of the Methanococcus jannaschii MJ1256 gene has been expressed in Escherichia coli, purified to homogeneity, and shown to be involved in coenzyme F420 biosynthesis. The protein catalyzes the transfer of the 2-phospholactate moiety from lactyl (2) diphospho-(5')guanosine (LPPG) to 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo) with the formation of the L-lactyl phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F420-0) and GMP. On the basis of the reaction catalyzed, the enzyme is named LPPG:Fo 2-phospho-L-lactate transferase. Since the reaction is the fourth step in the biosynthesis of coenzyme F420, the enzyme has been designated as CofD, the product of the cofD gene. The transferase requires Mg2+ for activity, and the catalysis does not appear to proceed via a covalent intermediate. To a lesser extent CofD also catalyzes a no. of addnl. reactions that include the formation of Fo-P, when the enzyme is incubated with Fo and GDP, GTP, pyrophosphate, or tripolyphosphate, and the hydrolysis of F420-0 to Fo. All of these side reactions can be rationalized as occurring by a common mechanism. CofD has no recognized sequence similarity to any previously characterized enzyme.
15. 15

    Bashiri, G., Antoney, J., Jirgis, E. N. M., Shah, M. V., Ney, B., Copp, J., Stuteley, S. M., Sreebhavan, S., Palmer, B., Middleditch, M., Tokuriki, N., Greening, C., Scott, C., Baker, E. N., and Jackson, C. J. (2019) A revised biosynthetic pathway for the cofactor F₄₂₀ in prokaryotes. Nat. Commun. 10, 1558,  DOI: 10.1038/s41467-019-09534-x

    Google Scholar

    15

    A revised biosynthetic pathway for the cofactor F420 in prokaryotes

    Bashiri Ghader; Jirgis Ehab N M; Stuteley Stephanie M; Middleditch Martin; Baker Edward N; Antoney James; Shah Mihir V; Ney Blair; Greening Chris; Scott Colin; Jackson Colin J; Antoney James; Ney Blair; Jackson Colin J; Copp Janine; Tokuriki Nobuhiko; Sreebhavan Sreevalsan; Palmer Brian; Greening Chris

    Nature communications (2019), 10 (1), 1558 ISSN:.

    Cofactor F420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F420 has not been fully elucidated: neither the enzyme that generates the putative intermediate 2-phospho-L-lactate, nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we present the structure of the guanylyltransferase FbiD and show that, along with its archaeal homolog CofC, it accepts phosphoenolpyruvate, rather than 2-phospho-L-lactate, as the substrate, leading to the formation of the previously uncharacterized intermediate dehydro-F420-0. The C-terminal domain of FbiB then utilizes FMNH2 to reduce dehydro-F420-0, which produces mature F420 species when combined with the γ-glutamyl ligase activity of the N-terminal domain. These new insights have allowed the heterologous production of F420 from a recombinant F420 biosynthetic pathway in Escherichia coli.
16. 16

    Bashiri, G., Rehan, A. M., Sreebhavan, S., Baker, H. M., Baker, E. N., and Squire, C. J. (2016) Elongation of the poly-gamma-glutamate tail of F₄₂₀ requires both domains of the F420:gamma-glutamyl ligase (FbiB) of Mycobacterium tuberculosis. J. Biol. Chem. 291, 6882– 6894,  DOI: 10.1074/jbc.M115.689026

    Google Scholar

    16

    Elongation of the poly-γ-glutamate tail of F420 requires both domains of the F420:γ-glutamyl ligase (FbiB) of Mycobacterium tuberculosis

    Bashiri, Ghader; Rehan, Aisyah M.; Sreebhavan, Sreevalsan; Baker, Heather M.; Baker, Edward N.; Squire, Christopher J.

    Journal of Biological Chemistry (2016), 291 (13), 6882-6894CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)

    Coenzyme F420 is an electron carrier with a major role in the oxidoreductive reactions of M. tuberculosis, the causative agent of tuberculosis. A γ-glutamyl ligase catalyzes the final steps of the F420 biosynthesis pathway by successive addns. of L-glutamate residues to F420-0, producing a poly-γ-glutamate tail. The enzyme responsible for this reaction in archaea (CofE) comprises a single domain and produces F420-2 as the major species. The homologous M. tuberculosis enzyme, FbiB, is a 2-domain protein and produces F420 with predominantly 5-7 L-glutamate residues in the poly-γ-glutamate tail. The N-terminal domain of FbiB is homologous to CofE with an annotated γ-glutamyl ligase activity, whereas the C-terminal domain has sequence similarity to an FMN-dependent family of nitroreductase enzymes. Here, the authors demonstrate that full-length FbiB adds multiple L-glutamate residues to F420-0 in vitro to produce F420-5 after 24 h; communication between the 2 domains is crit. for full γ-glutamyl ligase activity. The authors also present crystal structures of the C-terminal domain of FbiB in apo-, F420-0-, and FMN-bound states, displaying distinct sites for F420-0 and FMN ligands that partially overlap. Finally, the authors discuss the features of a full-length structural model produced by SAXS and its implications for the role of N- and C-terminal domains in catalysis.
17. 17

    Li, H., Graupner, M., Xu, H., and White, R. H. (2003) CofE catalyzes the addition of two glutamates to F₄₂₀-0 in F₄₂₀ coenzyme biosynthesis in Methanococcus jannaschii. Biochemistry 42, 9771– 9778,  DOI: 10.1021/bi034779b

    Google Scholar

    17

    CofE Catalyzes the Addition of Two Glutamates to F420-0 in F420 Coenzyme Biosynthesis in Methanococcus jannaschii

    Li, Hong; Graupner, Marion; Xu, Huimin; White, Robert H.

    Biochemistry (2003), 42 (32), 9771-9778CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

    The protein product of the Methanococcus jannaschii MJ0768 gene has been expressed in Escherichia coli, purified to homogeneity, and shown to catalyze the GTP-dependent addn. of two L-glutamates to the L-lactyl phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F420-0) to form F420-0-glutamyl-glutamate (F420-2). Since the reaction is the fifth step in the biosynthesis of coenzyme F420, the enzyme has been designated as CofE, the product of the cofE gene. Gel filtration chromatog. indicates CofE is a dimer. The enzyme has no recognized sequence similarity to any previously characterized proteins. The enzyme has an abs. requirement for a divalent metal ion and a monovalent cation. Among the metal ions tested, a mixt. of Mn2+, Mg2+, and K+ is the most effective. CofE catalyzes amide bond formation with the cleavage of GTP to GDP and inorg. phosphate, likely involving the activation of the free carboxylate group of F420-0 to give an acyl phosphate intermediate. Evidence for the occurrence of this intermediate is presented. A reaction mechanism for the enzyme is proposed and compared with other members of the ADP-forming amide bond ligase family.
18. 18

    Selengut, J. D. and Haft, D. H. (2010) Unexpected abundance of coenzyme F(420)-dependent enzymes in Mycobacterium tuberculosis and other actinobacteria. J. Bacteriol. 192, 5788– 5798,  DOI: 10.1128/JB.00425-10

    Google Scholar

    18

    Unexpected abundance of coenzyme F420-dependent enzymes in Mycobacterium tuberculosis and other actinobacteria

    Selengut, Jeremy D.; Haft, Daniel H.

    Journal of Bacteriology (2010), 192 (21), 5788-5798CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)

    Regimens targeting Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), require long courses of treatment and a combination of three or more drugs. An increase in drug-resistant strains of M. tuberculosis demonstrates the need for addnl. TB-specific drugs. A notable feature of M. tuberculosis is coenzyme F420, which is distributed sporadically and sparsely among prokaryotes. This distribution allows for comparative genomics-based investigations. Phylogenetic profiling (comparison of differential gene content) based on F420 biosynthesis nominated many actinobacterial proteins as candidate F420-dependent enzymes. Three such families dominated the results: the luciferase-like monooxygenase (LLM), pyridoxamine 5'-phosphate oxidase (PPOX), and deazaflavin-dependent nitroreductase (DDN) families. The DDN family was detd. to be limited to F420-producing species. The LLM and PPOX families were obsd. in F420-producing species as well as species lacking F420 but were particularly numerous in many actinobacterial species, including M. tuberculosis. Partitioning the LLM and PPOX families based on an organism's ability to make F420 allowed the application of the SIMBAL (sites inferred by metabolic background assertion labeling) profiling method to identify F420-correlated subsequences. These regions were found to correspond to flavonoid cofactor binding sites. Significantly, these results showed that M. tuberculosis carries at least 28 sep. F420-dependent enzymes, most of unknown function, and a paucity of FMN-dependent proteins in these families. While prevalent in mycobacteria, markers of F420 biosynthesis appeared to be absent from the normal human gut flora. These findings suggest that M. tuberculosis relies heavily on coenzyme F420 for its redox reactions. This dependence and the cofactor's rarity may make F420-related proteins promising drug targets.
19. 19

    Ney, B., Ahmed, F. H., Carere, C. R., Biswas, A., Warden, A. C., Morales, S. E., Pandey, G., Watt, S. J., Oakeshott, J. G., Taylor, M. C., Stott, M. B., Jackson, C. J., and Greening, C. (2017) The methanogenic redox cofactor F₄₂₀ is widely synthesized by aerobic soil bacteria. ISME J. 11, 125– 137,  DOI: 10.1038/ismej.2016.100

    Google Scholar

    19

    The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria

    Ney, Blair; Ahmed, F. Hafna; Carere, Carlo R.; Biswas, Ambarish; Warden, Andrew C.; Morales, Sergio E.; Pandey, Gunjan; Watt, Stephen J.; Oakeshott, John G.; Taylor, Matthew C.; Stott, Matthew B.; Jackson, Colin J.; Greening, Chris

    ISME Journal (2017), 11 (1), 125-137CODEN: IJSOCF; ISSN:1751-7362. (Nature Publishing Group)

    F420 is a low-potential redox cofactor that mediates the transformations of a wide range of complex org. compds. Considered one of the rarest cofactors in biol., F420 is best known for its role in methanogenesis and has only been chem. identified in two phyla to date, the Euryarchaeota and Actinobacteria. In this work, we show that this cofactor is more widely distributed than previously reported. We detected the genes encoding all five known F420 biosynthesis enzymes (cofC, cofD, cofE, cofG and cofH) in at least 653 bacterial and 173 archaeal species, including members of the dominant soil phyla Proteobacteria, Chloroflexi and Firmicutes. Metagenome datamining validated that these genes were disproportionately abundant in aerated soils compared with other ecosystems. We confirmed through high-performance liq. chromatog. anal. that aerobically grown stationary-phase cultures of three bacterial species, Paracoccus denitrificans, Oligotropha carboxidovorans and Thermomicrobium roseum, synthesized F420, with oligoglutamate sidechains of different lengths. To understand the evolution of F420 biosynthesis, we also analyzed the distribution, phylogeny and genetic organization of the cof genes. Our data suggest that although the Fo precursor to F420 originated in methanogens, F420 itself was first synthesized in an ancestral actinobacterium. F420 biosynthesis genes were then disseminated horizontally to archaea and other bacteria. Together, our findings suggest that the cofactor is more significant in aerobic bacterial metab. and soil ecosystem compn. than previously thought. The cofactor may confer several competitive advantages for aerobic soil bacteria by mediating their central metabolic processes and broadening the range of org. compds. they can synthesize, detoxify and mineralize.
20. 20

    Eirich, L. D., Vogels, G. D., and Wolfe, R. S. (1978) Proposed structure for coenzyme F₄₂₀ from Methanobacterium. Biochemistry 17, 4583– 4593,  DOI: 10.1021/bi00615a002

    Google Scholar

    20

    Proposed structure for coenzyme F420 from methanobacterium

    Eirich, L. Dudley; Vogels, Godfried D.; Wolfe, Ralph S.

    Biochemistry (1978), 17 (22), 4583-93CODEN: BICHAW; ISSN:0006-2960.

    The low-potential electron carrier, coenzyme F420, was purified from Methanobacterium strain M.o.H. A yield of 1.60 mg/kg of wet-packed cells was obtained. The results of anal. of hydrolytic fragments and periodate oxidn. products of the coenzyme, by IR, UV-visible, 1H and 13C NMR spectrometry, mass spectrometry, and quant. elemental analyses indicate that coenzyme F420 is: N,[N-[O-[5-(8-hydroxy-5-deazaisoalloxazin-10-yl)-2,3,4-trihydroxy-4-pentoxyhydroxyphosphinyl]-L-lactyl]-γ-L-glutamyl]-L-glutamic acid. A convenient trivial name would be the N-(N-L-lactyl-γ-L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5'-phosphate. Proof of structure by org. synthesis was not performed; the stereochem. configuration of the hydroxyl groups on the side chain as well as the position of the hydroxyl group on the arom. ring require confirmation by org. synthesis of the mol.
21. 21

    Cheeseman, P., Toms-Wood, A., and Wolfe, R. S. (1972) Isolation and properties of a fluorescent compound, factor 420, from Methanobacterium strain M.o.H. J. Bacteriol. 112, 527– 531

    Google Scholar

    21

    Isolation and properties of a fluorescent compound, factor420, from Methanobacterium strain M.o.H

    Cheeseman, P.; Toms-Wood, A.; Wolfe, R. S.

    Journal of Bacteriology (1972), 112 (1), 527-31CODEN: JOBAAY; ISSN:0021-9193.

    A new fluorescent compd. factor420 (F420), which is involved in the H metabolism of H-grown Methanobacterium strain M.o.H. has been isolated and purified. Acid hydrolysis of this compd. with 6M HCl for 24 hr releases a ninhydrin-pos. compd. (glutamic acid), an acid-stable chromophore, phosphate, and an ether-sol. phenolic component. F420 may be reduced by Na dithionite or Na borohydride at pH 7.3 with concomitant loss of its fluorescence and its major absorption peak at 420 nm. Crude cell-free exts. of strain M.o.H. reduce F420 only under a H atm. F420 is photolabile aerobically in neutral and basic solns., whereas the acid-stable chromophore is not photolabile under these conditions. An approx. mol. wt. of 630 ± 8% for F420 was detd. by Sephadex G-25 chromatog. At the present time, F420 is proposed as a trivial name for the unknown fluorescent compd. because of its strong absorption max. of 420 nm at pH 7.
22. 22

    Lackner, G., Peters, E. E., Helfrich, E. J., and Piel, J. (2017) Insights into the lifestyle of uncultured bacterial natural product factories associated with marine sponges. Proc. Natl. Acad. Sci. U. S. A. 114, E347– E356,  DOI: 10.1073/pnas.1616234114

    Google Scholar

    22

    Insights into the lifestyle of uncultured bacterial natural product factories associated with marine sponges

    Lackner, Gerald; Peters, Eike Edzard; Helfrich, Eric J. N.; Piel, Jorn

    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (3), E347-E356CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

    The as-yet uncultured filamentous bacteria Candidatus Entotheonella factor and Candidatus Entotheonella gemina live assocd. with the marine sponge Theonella swinhoei Y, the source of numerous unusual bioactive natural products. Belonging to the proposed candidate phylum Tectomicrobia, Candidatus Entotheonella members are only distantly related to any cultivated organism. The Ca. E. factor has been identified as the source of almost all polyketide and modified peptides families reported from the sponge host, and both Ca. Entotheonella phylotypes contain numerous addnl. genes for as-yet unknown metabolites. Here, we provide insights into the biol. of these remarkable bacteria using genomic, (meta)proteomic, and chem. methods. The data suggest a metabolic model of Ca. Entotheonella as facultative anaerobic, organotrophic organisms with the ability to use methanol as an energy source. The symbionts appear to be auxotrophic for some vitamins, but have the potential to produce most amino acids as well as rare cofactors like coenzyme F420. The latter likely accounts for the strong autofluorescence of Ca. Entotheonella filaments. A large expansion of protein families involved in regulation and conversion of org. mols. indicates roles in host-bacterial interaction. In addn., a massive overrepresentation of members of the luciferase-like monooxygenase superfamily points toward an important role of these proteins in Ca. Entotheonella. Furthermore, we performed mass spectrometric imaging combined with fluorescence in situ hybridization to localize Ca. Entotheonella and some of the bioactive natural products in the sponge tissue. These metabolic insights into a new candidate phylum offer hints on the targeted cultivation of the chem. most prolific microorganisms known from microbial dark matter.
23. 23

    Lackner, G., Moebius, N., Partida-Martinez, L. P., Boland, S., and Hertweck, C. (2011) Evolution of an endofungal lifestyle: deductions from the Burkholderia rhizoxinica genome. BMC Genomics 12, 210,  DOI: 10.1186/1471-2164-12-210

    Google Scholar

    23

    Evolution of an endofungal lifestyle: deductions from the Burkholderia rhizoxinica genome

    Lackner, Gerald; Moebius, Nadine; Partida-Martinez, Laila P.; Boland, Sebastian; Hertweck, Christian

    BMC Genomics (2011), 12 (), 210CODEN: BGMEET; ISSN:1471-2164. (BioMed Central Ltd.)

    Burkholderia rhizoxinica is an intracellular symbiont of the phytopathogenic zygomycete Rhizopus microsporus, the causative agent of rice seedling blight. The endosymbiont produces the antimitotic macrolide rhizoxin for its host. It is vertically transmitted within vegetative spores and is essential for spore formation of the fungus. To shed light on the evolution and genetic potential of this model organism, the whole genome of B. rhizoxinica HKI 0454 - a type strain of endofungal Burkholderia species - was analyzed. The genome consists of a structurally conserved chromosome and 2 plasmids. Compared to free-living Burkholderia species, the genome is smaller in size and harbors fewer transcriptional regulator genes. Instead, accumulation of transposons was obsd. over the genome. Prediction of primary metabolic pathways and transporters suggests that endosymbionts consume host metabolites like citrate, but might deliver some amino acids and cofactors to the host. The rhizoxin biosynthesis gene cluster shows evolutionary traces of horizontal gene transfer. Furthermore, gene clusters coding for nonribosomal peptide synthetases (NRPS) were analyzed. Notably, B. rhizoxinica lacks common genes which are dedicated to quorum sensing systems, but is equipped with a large no. of virulence-related factors and putative type III effectors. In conclusion, B. rhizoxinica is the first endofungal bacterium, whose genome has been sequenced. Models of evolution, metab., and tools are discussed for host-symbiont interaction of the endofungal bacterium deduced from whole genome analyses. Genome size and structure suggest that B. rhizoxinica is in an early phase of adaptation to the intracellular lifestyle (genome in transition). Anal. of tranporters and metabolic pathways allowed prediction of how metabolites might be exchanged between the symbiont and its host. Gene clusters for biosynthesis of secondary metabolites represent novel targets for genomic mining of cryptic natural products. In silico analyses of virulence-assocd. genes, secreted proteins, and effectors might inspire future studies on mol. mechanisms underlying bacterial-fungal interaction. The complete genome sequence with 3870 annotated proteins is deposited in GenBank/EMBL/DDBJ with accession nos. FR687359 (chromosome), FR687360 (plasmid pBRH01), and FR687361 (pBRH02).
24. 24

    Lackner, G. and Hertweck, C. (2011) Impact of endofungal bacteria on infection biology, food safety, and drug development. PLoS Pathog. 7, e1002096  DOI: 10.1371/journal.ppat.1002096

    Google Scholar

    24

    Impact of endofungal bacteria on infection biology, food safety, and drug development

    Lackner, Gerald; Hertweck, Christian

    PLoS Pathogens (2011), 7 (6), e1002096CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)

    There is no expanded citation for this reference.
25. 25

    Scherlach, K., Busch, B., Lackner, G., Paszkowski, U., and Hertweck, C. (2012) Symbiotic cooperation in the biosynthesis of a phytotoxin. Angew. Chem., Int. Ed. 51, 9615– 9618,  DOI: 10.1002/anie.201204540

    Google Scholar

    25

    Symbiotic Cooperation in the Biosynthesis of a Phytotoxin

    Scherlach, Kirstin; Busch, Benjamin; Lackner, Gerald; Paszkowski, Uta; Hertweck, Christian

    Angewandte Chemie, International Edition (2012), 51 (38), 9615-9618, S9615/1-S9615/5CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)

    Through a combination of genetic and chem. analyses we have solved the riddle of dual epoxidn. in rhizoxin biosynthesis. Sequencing and comparison of rhizoxin biosynthesis gene clusters and engineering of a mutant producing didesepoxy variants of rhizoxin reveled that the macrolide is first epoxidized by the cytochrome P 450 monoxygenase RhiH. By whole-cell transformation and cross-infection expts. we could unequivocally demonstrate that the 2,3-oxirane ring is introduced by the fungal host to specifically tailor the rhizoxin scaffold. According to rice seedling swelling assays, the addnl. epoxide moiety substantially increases phytotoxic potency. From an ecol. point of view, this finding is fully plausible, since the second epoxidn. is a specific trait of fungi belonging to the clade of rice seedling blight fungi. We therefore report for the first time on symbiotic synergism in biosynthesis of a secondary metabolite that has biol. significance.
26. 26

    Kiener, A., Orme-Johnson, W. H., and Walsh, C. T. (1988) Reversible conversion of coenzyme F₄₂₀ to the 8-OH-AMP and 8-OH-GMP esters, F₃₉₀-A and F₃₉₀-G, on oxygen exposure and reestablishment of anaerobiosis in Methanobacterium thermoautotrophicum. Arch. Microbiol. 150, 249– 253,  DOI: 10.1007/BF00407788

    Google Scholar

    26

    Reversible conversion of coenzyme F420 to the 8-OH-AMP and 8-OH-GMP esters, F390-A and F390-G, on oxygen exposure and reestablishment of anaerobiosis in Methanobacterium thermoautotrophicum

    Kiener, Andreas; Orme-Johnson, William H.; Walsh, Christopher T.

    Archives of Microbiology (1988), 150 (3), 249-53CODEN: AMICCW; ISSN:0302-8933.

    Intracellular levels of F390 (AMP and GMP adducts of the 5-deazaflavin cofactor F420) in M. thermoautotrophicum were analyzed after gasing fermenter cultures with several consecutive cycles of substrate gas and gas mixts. contg. 5% oxygen. No F390 was detected in growing cells, hydrogen-starved cells, and CO2-starved cells prior to O2 contamination. Also, no F390 was found in hydrogen-depleted cells after O2 treatment. Exposure of exponentially growing cells and CO2-starved cells to oxygen lead to the formation of F390 species; the increase in the detected amt. of F390 was coupled to a decrease of the F420 level. As soon as anaerobiosis was reestablished, F390 cofactors were degraded and growth proceeded. Independent of the physiol. condition of M. thermoautotrophicum, methanopterin was formed upon O2 exposure. After normal growth conditions were restored, the level of detected methanopterin decreased again.
27. 27

    Vermeij, P., Pennings, J. L., Maassen, S. M., Keltjens, J. T., and Vogels, G. D. (1997) Cellular levels of factor 390 and methanogenic enzymes during growth of Methanobacterium thermoautotrophicum deltaH. J. Bacteriol. 179, 6640– 6648,  DOI: 10.1128/jb.179.21.6640-6648.1997

    Google Scholar

    27

    Cellular levels of factor 390 and methanogenic enzymes during growth of Methanobacterium thermoautotrophicum ΔH

    Vermeij, Paul; Pennings, Jeroen L. A.; Maassen, Sander M.; Keltjens, Jan T.; Vogels, Godfried D.

    Journal of Bacteriology (1997), 179 (21), 6640-6648CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)

    Methanobacterium thermoautotrophicum ΔH was grown in a fed-batch fermentor and in a chemostat under a variety of 80% hydrogen-20% CO2 gassing regimes. During growth or after the establishment of steady-state conditions, the cells were analyzed for the content of adenylylated coenzyme F420 (factor F390-A) and other methanogenic cofactors. In addn., cells collected from the chemostat were measured for Me coenzyme M reductase isoenzyme (MCR I and MCR II) content as well as for specific activities of coenzyme F420-dependent and H2-dependent methylenetetrahydromethanopterin dehydrogenase (F420-MDH and H2-MDH, resp.), total (viologen-reducing) and coenzyme F420-reducing hydrogenase (FRH), factor F390 synthetase, and factor F390 hydrolase. The expts. were performed to investigate how the intracellular F390 concns. changed with the growth conditions used and how the variations were related to changes in levels of enzymes that are known to be differentially expressed. The levels of factor F390 varied in a way that is consistently understood from the biochem. mechanisms underlying its synthesis and degrdn. Moreover, a remarkable correlation was obsd. between expression levels of MCR I and II, F420-MDH, and H2-MDH and the cellular contents of the factor. These results suggest that factor F390 is a reporter compd. for hydrogen limitation and may act as a response regulator of methanogenic metab.
28. 28

    Newton, G. L., Buchmeier, N., and Fahey, R. C. (2008) Biosynthesis and functions of mycothiol, the unique protective thiol of Actinobacteria. Microbiol Mol. Biol. Rev. 72, 471– 494,  DOI: 10.1128/MMBR.00008-08

    Google Scholar

    28

    Biosynthesis and functions of mycothiol, the unique protective thiol of Actinobacteria

    Newton, Gerald L.; Buchmeier, Nancy; Fahey, Robert C.

    Microbiology and Molecular Biology Reviews (2008), 72 (3), 471-494CODEN: MMBRF7; ISSN:1092-2172. (American Society for Microbiology)

    A review. Mycothiol (MSH; AcCys-GlcN-Ins) is the major thiol found in Actinobacteria and has many of the functions of glutathione, which is the dominant thiol in other bacteria and eukaryotes but is absent in Actinobacteria. MSH functions as a protected reserve of cysteine and in the detoxification of alkylating agents, reactive oxygen and nitrogen species, and antibiotics. MSH also acts as a thiol buffer which is important in maintaining the highly reducing environment within the cell and protecting against disulfide stress. The pathway of MSH biosynthesis involves prodn. of GlcNAc-Ins-P by MSH glycosyltransferase (MshA), dephosphorylation by the MSH phosphatase MshA2 (not yet identified), deacetylation by MshB to produce GlcN-Ins, linkage to Cys by the MSH ligase MshC, and acetylation by MSH synthase (MshD), yielding MSH. Studies of MSH mutants have shown that the MSH glycosyltransferase MshA and the MSH ligase MshC are required for MSH prodn., whereas mutants in the MSH deacetylase MshB and the acetyltransferase (MSH synthase) MshD produce some MSH and/or a closely related thiol. Current evidence indicates that MSH biosynthesis is controlled by transcriptional regulation mediated by σB and σR in Streptomyces coelicolor. Identified enzymes of MSH metab. include mycothione reductase (disulfide reductase; Mtr), the S-nitrosomycothiol reductase MscR, the MSH S-conjugate amidase Mca, and an MSH-dependent maleylpyruvate isomerase. Mca cleaves MSH S-conjugates to generate mercapturic acids (AcCySR), excreted from the cell, and GlcN-Ins, used for resynthesis of MSH. The phenotypes of MSH-deficient mutants indicate the occurrence of one or more MSH-dependent S-transferases, peroxidases, and mycoredoxins, which are important targets for future studies. Current evidence suggests that several MSH biosynthetic and metabolic enzymes are potential targets for drugs against tuberculosis. The functions of MSH in antibiotic-producing streptomycetes and in bioremediation are areas for future study.
29. 29

    Zhang, L., Loh, K. C., Lim, J. W., and Zhang, J. X. (2019) Bioinformatics analysis of metagenomics data of biogas-producing microbial communities in anaerobic digesters: A review. Renewable Sustainable Energy Rev. 100, 110– 126,  DOI: 10.1016/j.rser.2018.10.021

    Google Scholar

    29

    Bioinformatics analysis of metagenomics data of biogas-producing microbial communities in anaerobic digesters: A review

    Zhang, Le; Loh, Kai-Chee; Lim, Jun Wei; Zhang, Jingxin

    Renewable & Sustainable Energy Reviews (2019), 100 (), 110-126CODEN: RSERFH; ISSN:1364-0321. (Elsevier Ltd.)

    Complex microbial communities in anaerobic digestion (AD) system play a vital role in the prodn. of biogas. An in-depth understanding of the microbial compns., diversity/similarity, metabolic networks, functional gene patterns, and relations between biodiversity and system functions at the genome level could help to optimize microbial productivity and contribute to enhancement of AD process. The study of microbial communities has been revolutionized in recent years with the development of high-throughput sequencing technologies. Anal. of high-throughput sequencing data and a suitable bioinformatics anal. approach therefore plays a very crit. role in the investigation of microbial metagenome. The present article reviews the overall procedure of processing metagenomics data of microbial communities for revealing metagenomics characterization using bioinformatics approaches. This includes (1) introduction of application case summary, (2) DNA extn. and high-throughput pyrosequencing, (3) processing metagenomics data using function-based bioinformatics platforms and tools, and (4) several specific bioinformatics anal. of anaerobic microbial communities. Key findings on anaerobic digestion via bioinformatics anal. are summarized. Limitations and future potential of bioinformatics approaches for anal. of metagenomics information of microbial communities are also discussed, with the hope of promoting its further development. Finally, a big-data-based precision fermn. platform using artificial neural network is proposed for integrating the bioinformatics data of microbial communities with performance of anaerobic digesters to facilitate the usage of huge metagenomics data.
30. 30

    Graupner, M. and White, R. H. (2001) Biosynthesis of the phosphodiester bond in coenzyme F(420) in the methanoarchaea. Biochemistry 40, 10859– 10872,  DOI: 10.1021/bi0107703

    Google Scholar

    30

    Biosynthesis of the Phosphodiester Bond in Coenzyme F420 in the Methanoarchaea

    Graupner, Marion; White, Robert H.

    Biochemistry (2001), 40 (36), 10859-10872CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

    The biochem. route for the formation of the phosphodiester bond in coenzyme F420, one of the methanogenic coenzymes, has been established in the methanoarchaea Methanosarcina thermophila and Methanococcus jannaschii. The first step in the formation of this portion of the F420 structure is the GTP-dependent phosphorylation of L-lactate to 2-phospho-L-lactate and GDP. The 2-phospho-L-lactate represents a new natural product that was chem. identified in Methanobacterium thermoautotrophicum, M. thermophila, and Mc. jannaschii. Incubation of cell exts. of both M. thermophila and Mc. jannaschii with [hydroxy-18O, carboxyl-18O2]lactate and GTP produced 2-phospho-L-lactate with the same 18O distribution as found in both the starting lactate and the lactate recovered from the incubation. These results indicate that the carboxyl oxygens are not involved in the phosphorylation reaction. Incubation of Sephadex G-25 purified cell exts. of M. thermophila or Mc. jannaschii with 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo), 2-phospho-L-lactate, and GTP or ATP lead to the formation of F420-0 (F420 with no glutamic acids). This transformation was shown to involve two steps: (i) the GTP- or ATP-dependent activation of 2-phospho-L-lactate to either lactyl(2)diphospho-(5')guanosine (LPPG) or lactyl(2)diphospho-(5')adenosine (LPPA) and (ii) the reaction of the resulting LPPG or LPPA with Fo to form F420-0 with release of GMP or AMP. Attempts to identify LPPG or LPPA intermediates by incubation of cell exts. with L-[U-14C]lactate, [U-14C]2-phospho-L-lactate, or [8-3H]GTP were not successful owing to the instability of these compds. toward hydrolysis. Synthetically prepd. LPPG and LPPA had half-lives of 10 min at 50° (at pH 7.0) and decompd. into GMP or AMP and 2-phospho-L-lactate via cyclic 2-phospho-L-lactate. No evidence for the functioning of the cyclic 2-phospho-L-lactate in the in vitro biosynthesis could be demonstrated. Incubation of cell exts. of M. thermophila or Mc. jannaschii with either LPPG or LPPA and Fo generated F420-0. In summary, this study demonstrates that the formation of the phosphodiester bond in coenzyme F420 follows a reaction scheme like that found in one of the steps of the DNA ligase reaction and in the biosynthesis of coenzyme B12 and phospholipids.
31. 31

    Warkentin, E., Mamat, B., Sordel-Klippert, M., Wicke, M., Thauer, R. K., Iwata, M., Iwata, S., Ermler, U., and Shima, S. (2001) Structures of F₄₂₀H₂: NADP⁺ oxidoreductase with and without its substrates bound. EMBO J. 20, 6561– 6569,  DOI: 10.1093/emboj/20.23.6561

    Google Scholar

    31

    Structures of F420H2:NADP+ oxidoreductase with and without its substrates bound

    Warkentin, Eberhard; Mamat, Bjorn; Sordel-Klippert, Melanie; Wicke, Michaela; Thauer, Rudolf K.; Iwata, Momi; Iwata, So; Ermler, Ulrich; Shima, Seigo

    EMBO Journal (2001), 20 (23), 6561-6569CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)

    Cofactor F420 is a 5'-deazaflavin deriv. first discovered in methanogenic archaea but later found also to be present in some bacteria. As a coenzyme, it is involved in hydride transfer reactions and as a prosthetic group in the DNA photolyase reaction. The authors report for the first time on the crystal structure of an F420-dependent oxidoreductase bound with F420. The structure of F420H2:NADP+ oxidoreductase resolved to 1.65 Å contains two domains: an N-terminal domain characteristic of a dinucleotide-binding Rossmann fold and a smaller C-terminal domain. The nicotinamide and the deazaflavin part of the two coenzymes are bound in the cleft between the domains such that the Si-faces of both face each other at a distance of 3.1 Å, which is optimal for hydride transfer. Comparison of the structures bound with and without substrates reveals that of the two substrates NADP has to bind first, the binding being assocd. with an induced fit.
32. 32

    Kunow, J., Schwörer, B., Stetter, K. O., and Thauer, R. K. (1993) A F(420)-dependent NADP reductase in the extremely thermophilic sulfate-reducing Archaeoglobus fulgidus. Arch. Microbiol. 160, 199– 205

    Google Scholar

    32

    A F420-dependent NADP reductase in the extremely thermophilic sulfate-reducing Archaeoglobus fulgidus

    Kunow, Jasper; Schwoerer, Beatrix; Stetter, Karl O.; Thauer, Rudolf K.

    Archives of Microbiology (1993), 160 (3), 199-205CODEN: AMICCW; ISSN:0302-8933.

    Archaeoglobus fulgidus, a sulfate-reducing Archaeon with a growth temp. optimum of 83°, uses the 5-deazaflavin coenzyme F420 rather than pyridine nucleotides in catabolic redox processes. The organism does, however, require reduced pyridine nucleotides for biosynthetic purposes. The authors describe here that the Archaeon contains a coenzyme F420-dependent NADP reductase which links anabolism to catabolism. The highly thermostable enzyme was purified 3600-fold by affinity chromatog. to apparent homogeneity in a 60% yield. The native enzyme with an apparent mol. mass of 55 kDa was composed of only one type of subunit of apparent mol. mass of 28 kDa. Spectroscopic anal. of the enzyme did not reveal the presence of any chromophoric prosthetic group. The purified enzyme catalyzed the reversible redn. of NADP (apparent KM 40 μM) with reduced F420 (apparent KM μM) with a specific activity of 660 U/mg (apparent Vmax) at pH 8.0 (pH optimum) and 80° (temp. optimum). It was specific for both coenzyme F420 and NADP. Stereochem. investigations showed that the F420-dependent NADP reductase was Si face specific with respect to C5 of F420 and Si face specific with respect to C4 of NADP.
33. 33

    Drenth, J., Trajkovic, M., and Fraaije, M. W. (2019) Chemoenzymatic synthesis of an unnatural deazaflavin cofactor that can fuel F-420-dependent enzymes. ACS Catal. 9, 6435– 6443,  DOI: 10.1021/acscatal.9b01506

    Google Scholar

    33

    Chemoenzymatic synthesis of an unnatural deazaflavin cofactor that can fuel F420-dependent enzymes

    Drenth, Jeroen; Trajkovic, Milos; Fraaije, Marco W.

    ACS Catalysis (2019), 9 (7), 6435-6443CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)

    F420-dependent enzymes are found in many microorganisms and can catalyze a wide range of redox reactions, including those with some substrates that are otherwise recalcitrant to enzyme-mediated redns. Unfortunately, the scarceness of the cofactor prevents application of these enzymes in biocatalysis. The best F420-producing organism, Mycobacterium smegmatis, only produces 1.4 μmol per L of culture. Therefore, we synthesized the unnatural cofactor FO-5'-phosphate, coined FOP. The FO core-structure was chem. synthesized, and an engineered riboflavin kinase from Corynebacterium ammoniagenes (CaRFK) was then used to phosphorylate the 5'-hydroxyl group. The triple F21H/F85H/A66I CaRFK mutant reached 80% of FO conversion in 12 h. The same enzyme could produce 1 mg (2.5 μmol) of FOP in 50 mL of reaction vol., which translates to a prodn. of 50 μmol/L. The activity toward FOP was tested for an enzyme of each of the three main structural classes of F420-dependent oxidoreductases. The sugar-6-phosphate dehydrogenase from Cryptosporangium arvum (FSD-Cryar), the F420:NADPH oxidoreductase from Thermobifida fusca (TfuFNO), and the F420-dependent reductases from Mycobacterium hassiacum (FDR-Mha) all showed activity for FOP. Although the activity for FOP was lower than that for F420, with slightly lower kcat and higher Km values, the catalytic efficiencies were only 2.0, 12.6, and 22.4 times lower for TfuFNO, FSD-Cryar, and FDR-Mha, resp. Thus, FOP could be a serious alternative for replacing F420 and might boost the application of F420-dependent enzymes in biocatalysis.
34. 34

    Ney, B., Carere, C. R., Sparling, R., Jirapanjawat, T., Stott, M. B., Jackson, C. J., Oakeshott, J. G., Warden, A. C., and Greening, C. (2017) Cofactor tail length modulates catalysis of bacterial F-420-dependent oxidoreductases. Front. Microbiol. 8, 1902,  DOI: 10.3389/fmicb.2017.01902

    Google Scholar

    34

    Cofactor Tail Length Modulates Catalysis of Bacterial F420-Dependent Oxidoreductases

    Ney Blair; Jirapanjawat Thanavit; Greening Chris; Ney Blair; Oakeshott John G; Warden Andrew C; Greening Chris; Carere Carlo R; Sparling Richard; Stott Matthew B; Sparling Richard; Jackson Colin J

    Frontiers in microbiology (2017), 8 (), 1902 ISSN:1664-302X.

    F420 is a microbial cofactor that mediates a wide range of physiologically important and industrially relevant redox reactions, including in methanogenesis and tetracycline biosynthesis. This deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F420 (two glutamates) from a methanogen isolate and long-chain F420 (five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F420 purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F420-dependent oxidoreductases. However, long-chain F420 bound to these enzymes with a six- to ten-fold higher affinity than short-chain F420. The cofactor side chain also significantly modulated the kinetics of the enzymes, with long-chain F420 increasing the substrate affinity (lower Km) but reducing the turnover rate (lower kcat) of the enzymes. Molecular dynamics simulations and comparative structural analysis suggest that the oligoglutamate chain of F420 makes dynamic electrostatic interactions with conserved surface residues of the oxidoreductases while the headgroup binds the catalytic site. In conjunction with the kinetic data, this suggests that electrostatic interactions made by the oligoglutamate tail result in higher-affinity, lower-turnover catalysis. Physiologically, we propose that bacteria have selected for long-chain F420 to better control cellular redox reactions despite tradeoffs in catalytic rate. Conversely, this suggests that industrial use of shorter-length F420 will greatly increase the rates of bioremediation and biocatalysis processes relying on purified F420-dependent oxidoreductases.
35. 35

    Jirapanjawat, T., Ney, B., Taylor, M. C., Warden, A. C., Afroze, S., Russell, R. J., Lee, B. M., Jackson, C. J., Oakeshott, J. G., Pandey, G., and Greening, C. (2016) The redox cofactor F₄₂₀ protects mycobacteria from diverse antimicrobial compounds and mediates a reductive detoxification system. Appl. Environ. Microbiol. 82, 6810– 6818,  DOI: 10.1128/AEM.02500-16

    Google Scholar

    35

    The redox cofactor F420 protects mycobacteria from diverse antimicrobial compounds and mediates a reductive detoxification system

    Jirapanjawat, Thanavit; Ney, Blair; Taylor, Matthew C.; Warden, Andrew C.; Afroze, Shahana; Russell, Robyn J.; Lee, Brendon M.; Jackson, Colin J.; Oakeshott, John G.; Pandey, Gunjan; Greening, Chris

    Applied and Environmental Microbiology (2016), 82 (23), 6810-6818CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)

    A defining feature of mycobacterial redox metab. is the use of an unusual deazaflavin cofactor, F420. This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including after antimicrobial treatment, although the mol. basis for this remains to be understood. In this work, we explored our hypothesis that F420 enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochem., and anal. chem. studies in relation to the model soil bacterium Mycobacterium smegmatis. Mutant strains unable to synthesize or reduce F420 were found to be more susceptible to a wide range of antibiotic and xenobiotic compds. Compds. from three classes of antimicrobial compds. traditionally resisted by mycobacteria inhibited the growth of F420 mutant strains at subnanomolar concns., namely, furanocoumarins (e.g., methoxsalen), arylmethanes (e.g., malachite green), and quinone analogs (e.g., menadione). We demonstrated that promiscuous F420H2-dependent reductases directly reduce these compds. by a mechanism consistent with hydride transfer. Moreover, M. smegmatis strains unable to make F420H2 lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compd. classes in whole-cell assays. In contrast, mutant strains were only slightly more susceptible to clin. antimycobacterials, and this appeared to be due to indirect effects of F420 loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F420 enhances antimicrobial resistance in mycobacteria and suggest that one function of the F420H2-dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify.
36. 36

    Mascotti, M. L., Kumar, H., Nguyen, Q. T., Ayub, M. J., and Fraaije, M. W. (2018) Reconstructing the evolutionary history of F-420-dependent dehydrogenases. Sci. Rep. 8, 17571,  DOI: 10.1038/s41598-018-35590-2

    Google Scholar

    36

    Reconstructing the evolutionary history of F420-dependent dehydrogenases

    Mascotti M Laura; Ayub Maximiliano Juri; Kumar Hemant; Nguyen Quoc-Thai; Fraaije Marco W; Nguyen Quoc-Thai; Nguyen Quoc-Thai

    Scientific reports (2018), 8 (1), 17571 ISSN:.

    During the last decade the number of characterized F420-dependent enzymes has significantly increased. Many of these deazaflavoproteins share a TIM-barrel fold and are structurally related to FMN-dependent luciferases and monooxygenases. In this work, we traced the origin and evolutionary history of the F420-dependent enzymes within the luciferase-like superfamily. By a thorough phylogenetic analysis we inferred that the F420-dependent enzymes emerged from a FMN-dependent common ancestor. Furthermore, the data show that during evolution, the family of deazaflavoproteins split into two well-defined groups of enzymes: the F420-dependent dehydrogenases and the F420-dependent reductases. By such event, the dehydrogenases specialized in generating the reduced deazaflavin cofactor, while the reductases employ the reduced F420 for catalysis. Particularly, we focused on investigating the dehydrogenase subfamily and demonstrated that this group diversified into three types of dehydrogenases: the already known F420-dependent glucose-6-phosphate dehydrogenases, the F420-dependent alcohol dehydrogenases, and the sugar-6-phosphate dehydrogenases that were identified in this study. By reconstructing and experimentally characterizing ancestral and extant representatives of F420-dependent dehydrogenases, their biochemical properties were investigated and compared. We propose an evolutionary path for the emergence and diversification of the TIM-barrel fold F420-dependent dehydrogenases subfamily.
37. 37

    Kumar, H., Nguyen, Q. T., Binda, C., Mattevi, A., and Fraaije, M. W. (2017) Isolation and characterization of a thermostable F_420:NADPH oxidoreductase from Thermobifida fusca. J. Biol. Chem. 292, 10123– 10130,  DOI: 10.1074/jbc.M117.787754

    Google Scholar

    37

    Isolation and characterization of a thermostable F420:NADPH oxidoreductase from Thermobifida fusca

    Kumar, Hemant; Nguyen, Quoc-Thai; Binda, Claudia; Mattevi, Andrea; Fraaije, Marco W.

    Journal of Biological Chemistry (2017), 292 (24), 10123-10130CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)

    F420H2-dependent enzymes reduce a wide range of substrates that are otherwise recalcitrant to enzyme-catalyzed redn., and their potential for applications in biocatalysis has attracted increasing attention. Thermobifida fusca is a moderately thermophilic bacterium and holds high biocatalytic potential as a source for several highly thermostable enzymes. We report here on the isolation and characterization of a thermostable F420: NADPH oxidoreductase (Tfu-FNO) from T. fusca, the first F420-dependent enzyme described from this bacterium. Tfu-FNO was heterologously expressed in Escherichia coli, yielding up to 200 mg of recombinant enzyme per L of culture. We found that Tfu-FNO is highly thermostable, reaching its highest activity at 65 °C and that Tfu-FNO is likely to act in vivo as an F420 reductase at the expense of NADPH, similar to its counterpart in Streptomyces griseus. We obtained the crystal structure of FNO in complex with NADP+ at 1.8 Å resoln., providing the first bacterial FNO structure. The overall architecture and NADP+-binding site of Tfu-FNO were highly similar to those of the Archaeoglobus fulgidus FNO (Af-FNO). The active site is located in a hydrophobic pocket between an N-terminal dinucleotide binding domain and a smaller C-terminal domain. Residues interacting with the 2'-phosphate of NADP+ were probed by targeted mutagenesis, indicating that Thr-28, Ser-50, Arg-51, and Arg-55 are important for discriminating between NADP+ and NAD+. Interestingly, a T28A mutant increased the kinetic efficiency >3-fold as compared with the wild-type enzyme when NADH is the substrate. The biochem. and structural data presented here provide crucial insights into the mol. recognition of the two cofactors, F420 and NAD(P)H by FNO.