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C-Diazeniumdiolate Graminine in the Siderophore Gramibactin Is Photoreactive and Originates from Arginine

  • Christina Makris
    Christina Makris
    Department of Chemistry & Biochemistry, University of California, Santa Barbara, California 93106-9510, United States
  • Jeffrey R. Carmichael
    Jeffrey R. Carmichael
    Department of Chemistry & Biochemistry, University of California, Santa Barbara, California 93106-9510, United States
  • Hongjun Zhou
    Hongjun Zhou
    Department of Chemistry & Biochemistry, University of California, Santa Barbara, California 93106-9510, United States
    More by Hongjun Zhou
  • , and 
  • Alison Butler*
    Alison Butler
    Department of Chemistry & Biochemistry, University of California, Santa Barbara, California 93106-9510, United States
    *Email: [email protected]
Cite this: ACS Chem. Biol. 2022, 17, 11, 3140–3147
Publication Date (Web):November 10, 2022
https://doi.org/10.1021/acschembio.2c00593

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

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Abstract

Siderophores are synthesized by microbes to facilitate iron acquisition required for growth. Catecholate, hydroxamate, and α-hydroxycarboxylate groups comprise well-established ligands coordinating Fe(III) in siderophores. Recently, a C-type diazeniumdiolate ligand in the newly identified amino acid graminine (Gra) was found in the siderophore gramibactin (Gbt) produced by Paraburkholderia graminis DSM 17151. The N–N bond in the diazeniumdiolate is a distinguishing feature of Gra, yet the origin and reactivity of this C-type diazeniumdiolate group has remained elusive until now. Here, we identify l-arginine as the direct precursor to l-Gra through the isotopic labeling of l-Arg, l-ornithine, and l-citrulline. Furthermore, these isotopic labeling studies establish that the N–N bond in Gra must be formed between the Nδ and Nω of the guanidinium group in l-Arg. We also show the diazeniumdiolate groups in apo-Gbt are photoreactive, with loss of nitric oxide (NO) and H+ from each d-Gra yielding E/Z oxime isomers in the photoproduct. With the loss of Gbt’s ability to chelate Fe(III) upon exposure to UV light, our results hint at this siderophore playing a larger ecological role. Not only are NO and oximes important in plant biology for communication and defense, but so too are NO-releasing compounds and oximes attractive in medicinal applications.

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Introduction

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A disproportionate sum of pharmaceuticals incorporate N–N bonds. Pharmaceuticals are often inspired by natural products, with at least 200 reported with an N–N bond. (1,2) From this subset of natural products, only a few harbor a C-type diazeniumdiolate functional group (Figure 1). (2)C-type diazeniumdiolates, like alanosine, (3,4) fragin, (5,6) and leudiazen, (7) are defined by the nitrosohydroxylamine group bonded to a carbon as opposed to N-type diazeniumdiolates, such as (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) (8) in which the nitrosohydroxylamine group is appended to a nitrogen atom (Figure 1). In 2018, the siderophore gramibactin (Gbt) was reported from Paraburkholderia graminis DSM 17151 with a new C-diazeniumdiolate amino acid, graminine (Gra; Figure 2). (9,10) Siderophores are small molecules produced by bacteria to facilitate the acquisition of iron, which is essential for microbial growth. (11) Gbt is comprised of six amino acids, which includes two d-Gra residues, along with Gly, d-allo Thr, l-Thr, and d-threo β-hydroxyAsp residues of which each Gra and β-hydroxyaspartic acid (β-OH-Asp) coordinate Fe(III). (12)

Figure 1

Figure 1. Selected N–N bonded compounds. C-diazeniumdiolate in alanosine, fragin, and leudiazen are shown in blue, the N-diazeniumdiolate in DETA NONOate is shown in red, and the N-nitrosourea in streptozocin is shown in green.

Figure 2

Figure 2. (a) Structure of Gbt with Gra in inset; (b) BGC of Gbt.

The biological signaling properties of nitric oxide (NO) are widespread in nature. Diazeniumdiolates, which are recognized as potential NO-donor compounds, are classified as C-, N-, O-, or S-type compounds among which the reactivity of NONOates has received the most attention. A particularly attractive feature of NONOates, which have potential clinical applications, (13−15) is their photoreactivity, producing two equivalents of NO. In contrast to NONOates, which have yet to be identified in natural products, little is known about the reactivity of C-type diazeniumdiolate natural products and the conditions under which NO may be released (Figure 1). (8,16)
The origin and biosynthesis of the N–N bond in natural products have attracted much interest, (1,17) in part due to the anticancer drug streptozocin (Zanosar, Figure 1) isolated from Streptomyces achromogenes var. streptozoticus NRRL 2697, which is effective against pancreatic cancers. (18,19) The enzyme SznF in the biosynthetic gene cluster (BGC) of streptozocin is reported to catalyze the formation of the N-nitrosourea group (Figure 1). (19) Prior to N–N bond formation, the heme-dioxygenase-like domain of SznF hydroxylates Nω-methyl-l-Arg at both the guanidinium Nδ and the unmethylated Nω positions. (19−21) Following hydroxylation, the cupin domain of SznF is proposed to catalyze the oxidative rearrangement of Nδ-hydroxy-Nω-hydroxy-Nω-methyl-l-Arg forming the N–N bond of streptozocin; however, the mechanism of N–N bond formation remains elusive. (19)
In the BGC for Gbt (Figure 2), the enzymes GrbE and GrbD have been identified through gene deletion studies to be essential for the formation of Gra. (10) GrbE shares sequence homology to several reported arginine hydroxylases, including AglA/AlpD and Mhr24, which are Nδ-hydroxylases, (22−24) and DcsA, which is a Nω-hydroxylase (Figure S1). (25) GrbD shares sequence homology to only the cupin domain in SznF (Figure S1). (19) The homologies of GrbE to the Arg hydroxylases and GrbD to one domain of SznF is suggestive of l-Arg as the precursor of l-Gra, yet direct experiments to investigate l-Arg and the source of l-Gra have not been reported.
Herein, we report that the Gra C-diazeniumdiolate groups in apo-Gbt are photoreactive, losing an equivalent of NO and H+ from each d-Gra residue. Through isotopic feeding studies, we demonstrate that Gra in Gbt originates from Arg. We further structurally characterize the Gbt photoproduct as a mixture of E/Z oxime isomers.

Results and Discussion

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Apo-Gbt Loses Two Equivalents of Nitric Oxide upon Photolysis

Upon irradiation of apo-Gbt (5 mM, 3-(N-morpholino)propane sulfonic acid (MOPS), pH 8) with UV light (e.g., λ 254 nm Hg(Ar) pen lamp and in sunlight), the characteristic pH 8 diazeniumdiolate absorption band at 248 nm disappears (Figure 3a). Mass spectrometric analysis of apo-Gbt, m/z 835.3 [M + H]+, shows two successive mass losses of 30, as well as a mass at m/z 888.3 [M – 2H + Fe]+ indicative of the Fe(III)-Gbt complex (Figure 3b). The observed mass losses of m/z 30 in the mass spectrum are characteristic of diazeniumdiolates, reflecting the lability of the N–N bond in the presence of ionization energy in the mass spectrometer. (10)

Figure 3

Figure 3. UV photolysis of apo-Gbt. (a) UV-absorption spectra of apo-Gbt (44 μM) as a function of the time of photolysis (253.7 nm bandpass filter; 0–6 h in 5 mM MOPS pH 8.0). From 0 to 1 h, spectra were obtained at 4 min intervals; from 1 to 2 h, spectra were obtained at 10 min intervals; and from 2 to 3 h, spectra were obtained at 20 min intervals. Inset showing the isosbestic point between time 0 and 1 h; (b) MS of apo-Gbt; (c) MS of the photoproduct of Gbt.

Analysis of the purified photoproduct by mass spectrometry shows a new mass at m/z 773.3 [M + H]+ (Figure 3c). The difference of 62 mass units from apo-Gbt at m/z 835.3 [M + H]+ is consistent with the loss of two equivalents each of NO and H+. An Fe(III) bound mass was not observed in the mass spectrometry (MS) of the photoproduct, indicating a change in the Fe(III) coordinating groups. The mass of the photoproduct of 15N-enriched Gbt shows a mass loss of m/z 64, consistent with the release of two equiv. each of 15NO and H+. To investigate the biological relevance of the photoreactivity, aliquots were taken directly from an actively growing culture of P. graminis DSM 17151, two of which were photolyzed with different UV light sources. One aliquot was irradiated with a Hg(Ar) pen lamp and the other with sunlight. Following irradiation, the cells were pelleted and the crude supernatant was analyzed by ultra-high performance liquid chromatography (UPLC-MS), revealing only the photoproduct m/z 773.3 [M + H]+ without any trace of apo-Gbt, m/z 835 (Figure S2). In contrast, an aliquot from the same culture which was not exposed to UV light did not contain any of the photoproduct (Figure S2). The results of the colorimetric Griess test, which detects nitrite, (26−28) are also consistent with the photoinduced release of NO with subsequent oxidation to NO2 (Figure S3). Thus, the UV and mass spectral changes, along with the Griess test results suggest the photoreaction leads to the loss of NO and a H+ from each of the two d-Gra residues in apo-Gbt.
The β-hydroxyaspartate coordinated to Fe(III) in Gbt is photoreactive, complicating the investigation of possible NO labilization while bound to Fe(III). (29,30) Analysis of the Fe(III)-Gbt photoproducts by UPLC-MS initially shows the molecular ion m/z 842 [M – 2H + Fe]+, corresponding to the loss of CO2 and two H+’s from the β-OH-Asp, but retaining the coordination to iron with the two diazeniumdiolate ligands (Figure S4). Initially, NO is not labilized in the photolysis of Fe(III)-Gbt; however, further mechanistic investigations of the photoreactivity are underway.
The λmax of the diazeniumdiolate in apo-Gbt is strongly pH dependent, shifting from 248 nm at pH 10 to 220 nm at pH 2. (9) Not surprisingly, we find the attendant photoreactivity of apo-Gbt at low pH, as measured by the disappearance of the 220 nm absorption peak, is reduced both on irradiation at 254 nm and in sunlight. The reduced photosensitivity at low pH reflects the poor overlap of the irradiating wavelength and blue-shift of the absorption band at pH 2 (λmax 220 nm) compared to pH 8 (λmax 248 nm).

Isotopic Labeling Establishes Gra Originates from Arg

To investigate the origin of Gra, we employed 13C and 15N isotopic feeding studies in the growth of P. graminis DSM 17151. When this strain is grown with 15NH4Cl as the sole nitrogen source, an isotopic mass for Gbt of M + 10 is observed (i.e., m/z 845.3 [M + H]+), consistent with 15N-enrichment at each nitrogen (Figure S5). However, when P. graminis DSM 17151 is cultured with both 15NH4Cl and 14N-l-Arg, an isotopic mass at m/z 839.3 [M + H]+ is observed for Gbt which is four mass units higher than that for Gbt (Figures 4 and S6). The isotopic mass of M + 4 is consistent with 15N incorporation into d-threo OH-Asp, l-Thr, d-allo Thr, and Gly, whereas the nitrogens in Gra are consistent with incorporation from the supplemented 14N-l-Arg (Figure 4c). MS analysis of the M + 4 Gbt derivative substantiates the incorporation of 14N in each Gra residue with two ion mass fragments of 30 (14NO) rather than 31 (15NO) (Figure 4a). Upon photolysis of the partially 15N-enriched apo-Gbt, a photoproduct with a mass of m/z 777.3 [M + H]+ is observed (Figures S6 and S7), also consistent with the incorporation of 14N Gra in the M + 4 Gbt derivative. While photolysis of fully 15N enriched Gbt leads to a photoproduct with a mass loss of 64, we observe a mass loss of 62 in this M + 4 Gbt derivative, consistent with 14N Gra (Figures S5 and S7).

Figure 4

Figure 4. Isotope feeding studies demonstrate l-Gra originates from l-Arg. (a) Gbt isolated from the P. graminis DSM 17151 culture grown on 15NH4Cl and 14N-Arg. Isotopic mass of Gbt at m/z 839 is present along with two mass losses of 14NO; (b) Gbt isolated from the P. graminis DSM 17151 culture grown on 14NH4Cl and 13C615N4-Arg. Isotopic mass of Gbt at m/z 851 is present along with two mass losses of 15NO; (c) Scheme depicting isotopic labeling of Gra originating from 14N-Arg or from 13C615N4-Arg.

To further probe the origin of Gra in Gbt, the P. graminis DSM 17151 culture medium was supplemented with 1 mM 13C15N-l-Arg. The resulting Gbt showed isotopic masses at m/z 851.3 (M + 16) and 852.3 (M + 17; Figure 4b), as well as 853.3 (M + 18). The M + 16 mass is expected based on the isotopic labeling of five 13C’s and three 15N’s per Gra (Figure 4c). The M + 17 and M + 18 Gbt likely result from the incorporation of an 15N released from 13C15N-l-Arg and then taken up in the biosynthesis of the other amino acids, as confirmed by nuclear magnetic resonance (NMR) (Figures S8–S20).
NMR characterization of Gbt purified from P. graminis DSM 17151 grown in the presence of 13C15N-Arg and dissolved in hexadeuterodimethyl sulfoxide (DMSO-d6) and D2O (1H, 13C, heteronuclear single quantum coherence-distortionless enhancement by polarization transfer (HSQC-DEPT), correlated spectroscopy (COSY), 1H–13C heteronuclear multiple bond correlation (HMBC), 13C–13C COSY, 1H–15N HMBC, and 13C incredible natural abundance double quantum transfer experiment (INADEQUATE)) confirms the incorporation of fully labeled 13C15N Gra residues in Gbt (Figures S8–S20). Connectivity of 13C-enriched Cα, Cβ, Cγ, and Cδ in each Gra residue was obtained by 13C INADEQUATE and 13C–13C COSY experiments (Figures S17 and S18). 1H NMR spectra show mixtures of 14N–H and 15N–1H coupled (J = 90 Hz) amides at each amino acid residue (Figure S10). (31) The 15N-labeling in Gly, each Thr and β-OH-Asp must result from 15NH3 released from 13C15N-Arg, either as a byproduct of arginase and the urea cycle, (32−36) or during the putative oxidative conversion of hydroxy-Arg to Gra, and the subsequent incorporation of 15NH3 in the biosynthesis of these amino acids. The M + 17 Gbt isotopic mass is consistent with the incorporation of one 15N-labeled amino acid at any of the four amino acids distinct from Gra (Figures S10 and S21). A less intense M + 18 isotopic mass is also observed (Figure 4b) suggestive of the incorporation of two 15NH3-labeled amino acids. Incorporation of 13C released by these processes is not detected in the isolated Gbt reflecting the higher concentration of other carbon sources in the growth medium.
It has been speculated that Gra originates from l-ornithine; (17) therefore we also investigated whether 14N-ornithine and 14N-citrulline added to the P. graminis DSM 17151 growth medium with 15NH4Cl are converted to Gra. l-Orn and l-Cit are both on the microbial biosynthetic route to l-Arg, (32−34) and therefore it may be more energetically favorable for the microbe to use the added 14N-l-Orn or 14N-l-Cit before initiating the de novo biosynthesis of l-Arg using the added 15NH4Cl (Figure S21). Gbt isolated from cultures supplemented with 14N-l-Orn show two primary isotopic masses of Gbt, m/z 841.3 [M + H]+ (i.e., M + 6) and m/z 843.3 [M + H]+ (i.e., M + 8), with attendant mass losses of 15NO present in the mass spectrum (Figure S22). 14N-l-Orn will yield l-Arg with both guanidinium 15Nω’s enriched. Both the peptidyl amide nitrogen and hydroxylamine nitrogen in the resulting Gra remain as naturally abundant 14N, while the nitroso nitrogen is 15N-enriched (Figure S22). In contrast to 14N-l-Orn, 14N-l-Cit would be expected to yield l-Arg with the two guanidium Nω’s existing as an equivalent mixture of 14N and 15N (Figure S23). When cultures were supplemented with 14N-l-Cit, a range of isotopic masses are observed for Gbt (Figure S23), consistent with Gra arising from distinct isotopically enriched l-Arg residues. The results of isotopic feeding of P. graminis DSM 17151 with 14N-l-Orn and 14N-l-Cit in the presence of 15NH4Cl are thus consistent with l-Gra originating from l-Arg and suggest the conversion is enzymatic. The observation of two distinct isotopic molecular ions resulting from 14N-l-Orn supplementation in contrast to an array of isotopic species with 14N-l-Cit supplementation demonstrates that the N–N bond must form between the 14Nδ of l-Arg, and either of the 15Nω in the guanidinium groups. Investigations into the mechanism of the oxidative conversion of Arg to Gra are in progress.

Photolysis of Isotopically Labeled Gbt Reveals the Photoproduct Is an Oxime

The 13C15N-isotopically enriched Gra residues in Gbt were also used to characterize the photoproduct of apo-Gbt, which could produce nitroso or oxime products (Figure 5). Immediately following the complete photolysis of apo-Gbt in a quartz NMR tube (pD 8.0, 6.2 mM phosphate buffer, 99.9% D2O), as indicated by the disappearance of the diazeniumdiolate absorption band at 248 nm, the reaction solution was evaluated by NMR (Figures 5 and S24–S29). The 1H (13C and 15N decoupled) and 13C NMR spectra show two new 1H resonances at 6.90 and 7.53 ppm and two new 13C resonances at 152.72 and 153.10 ppm not present in Gbt (Figures 5c and S24–S26). The 1H-13C HSQC spectrum of the photoproduct shows that the two new 1H resonances correlate to the two new 13C resonances (Figure 5c). The formation of an oxime is consistent with the Cδ of the Gra residues undergoing a change from sp3 to sp2 hybridization. Additionally, both E and Z oxime isomers are observed for both Gra residues, with the E isomer proton being shifted further downfield than the Z configuration. (37,38) The Cδ 1H and 13C shifts of both Gra residues in apo-Gbt (Figure 5a) disappear upon photolysis (Figure 5b). Coupling between 15N, 13C, and 1H nuclei on the 13C15N-enriched-Gra residues causes inconsistencies in the phasing, as can be observed at the Cα’s of the Gra residues, which are adjacent to a 15N and two 13C nuclei (Figures S11 and S16). HSQC resonances from the remaining amino acid residues in Gbt derive from the natural abundance of 13C. In comparison, the 1H–13C correlations of Cα, Cβ, Cγ, and Cδ in 13C-enriched Gra are apparent, including the disappearance of the 1H–13C correlation from each Gra Cδ in the photoproduct (Figure 5a,b). Full NMR characterization of the photoproduct shows the rest of the structure remains unchanged. A TOtal Correlated SpectroscopY (TOCSY) NMR experiment establishes that the E and Z oxime conformations are present in the complete 1H spin systems of both Gra1 and Gra2 photoproducts (Figure S30). Thus, the NMR results establish the photoproduct of apo-Gbt is a mixture of E/Z oxime isomers (Figure 5d). The ratio of the 1H resonance integrations of the E to Z oxime (1:1.7) immediately after photolysis shows the Z configuration is the dominant form. After 2 days at −20 °C, the peak integration (1:0.7) shows a shift to the E configuration, which has been reported as the more biologically active isomer. (39) The E/Z isomerization process is under further investigation.

Figure 5

Figure 5. Multiplicity edited 1H–13C HSQC NMR spectra show the formation of E and Z oxime isomers upon photolysis of 13C15N-Gra-enriched-Gbt (pD 8, 6.2 mM, Pi in 99.9% D2O). (a) 1H–13Cδ correlations in Gra residues of apo-Gbt at 4.20, 61.66 ppm and 4.22, 61.93 ppm; (b) region of HSQC showing disappearance of the 1H–13Cδ correlations in the photoproduct; (c) new downfield 1H–13C HSQC correlations in photolyzed 13C15N-Gra-enriched Gbt, with chemical shifts consistent with E/Z oxime isomers; (d) scheme showing release of NO from Gra yields E and Z oxime isomers but not a nitroso photoproduct. Spectra collected in D2O.

Conclusions

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Apo-Gbt is photoreactive, losing NO and H+ from each d-Gra upon irradiation with UV light (Figure 6). The downfield shift of the Gra Cδ 1H and 13C resonances in the 13C15N-enriched Gbt photoproduct is consistent with the formation of an oxime. The 0.6 ppm difference between the new 1H resonances (6.90, 7.53 ppm) is indicative of a mixture of E/Z oxime isomers. (37) The mass loss of 64 observed in the 13C15N-Gbt photoproduct is consistent with release of 15NO and H+ from each Gra. Thus, the photoreactivity of this C-diazeniumdiolate natural product is established, and is among the first examples of photorelease of NO from a C-diazeniumdiolate. Isotopic feeding of P. graminis DSM 17151 with 13C615N4l-Arg establishes that it is the precursor to l-Gra, with all 13C’s and 15N’s in Gra originating from the enriched-Arg. Moreover, we demonstrate with isotopic labeling that l-Orn and l-Cit are not direct precursors to l-Gra, with these results demonstrating that the N–N bond is formed between Nδ and Nω of the l-Arg guanidinium group. In ongoing research, we are investigating the role of GrbE in l-Arg hydroxylation and the role of GrbD in the oxidative rearrangement of hydroxy-arginine in the formation of the N–N bond in l-Gra.

Figure 6

Figure 6. Photolysis of 13C15N-Gra-enriched-Gbt yields E/Z oxime isomers.

Oximes are an established class of pharmaceuticals, with several FDA-approved oxime-based therapeutics used as antidotes for organophosphate poisoning, as well as in cephalosporin antibiotics. (40) In addition, oximes and NO are biologically active in plants, serving functions facilitating plant defense, communication, and growth. (39,41,42) While the pathways involved in NO signaling in response to stress are still largely unknown, NO has been shown to regulate gene expression and hormonal activity, as well as provide protection under oxidative stress by interacting with reactive oxygen species. (42) It is plausible that the photochemical release of NO and the formation of the oxime could be an unintended side reaction of Gbt, given oximes derived from amino acids commonly serve as intermediates to defensive compounds and plant hormones. (39) Free Gra could play a role in plant biology as a unique photoreactive precursor to produce an oxime and release NO when exposed to sunlight. Given that the C-diazeniumdiolate siderophores currently reported in the literature are all isolated from rhizospheric bacteria, Gbt may serve the dual purpose of Fe(III) acquisition, as well as providing a source of NO upon exposure to sunlight as part of a symbiotic relationship with the associated root nodules. (10)

Methods

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General Experimental Procedures

UV–Visible spectra were obtained on an Agilent Technologies Cary 300 UV–vis spectrometer. NMR spectroscopy was carried out at 25 °C on a Bruker 500 MHz spectrometer equipped with a Prodigy cold probe (1H, 13C, 1H–13C multiplicity edited HSQC, 1H–1H COSY, 13C–13C COSY, 1H–13C HMBC, 1H–15N HMBC, 13C INADEQUATE, and TOCSY) or on a Varian Inova 600 MHz spectrometer equipped with a 1H, 13C, 15N triple resonance inverse detection probe (1H with or without 13C, 15N, or 13C/15N decoupling, and 1H–15N HMBC with or without 13C decoupling). Spectra were collected in DMSO-d6 or D2O. Spectra were indirectly referenced by the residual solvent peak or 2H lock. MS analysis was carried out on a Waters Xevo G2-XS QTof with positive mode electrospray ionization coupled to an AQUITY UPLC H-Class system with a Waters BEH C18 column. Gbt samples were run with a linear gradient of 0 to 100% acetonitrile (0.1% formic acid) in ddH2O (0.1% formic acid) over 10 min. Deionized water was dispensed from a Milli-Q IQ 7000 water purification system (Resistivity 18.2 MΩ). All glassware was acid-washed with 4 M HCl and subsequently rinsed with Milli-Q water.

Bacterial Growth and Siderophore Isolation

P. graminis DSM 17151 was obtained from the German collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ). P. graminis DSM 17151 was maintained on Luria–Bertani (LB) agar plates at 30 °C. Single colonies were inoculated in 3 mL of LB media and grown for 24 h shaking at 30 °C, 180 rpm. The 3 mL starter cultures were used to inoculate 1 L cultures of iron-depleted MM9 medium (7 g K2HPO4, 2 g KH2PO4, 0.59 g NaCl, 1 g NH4Cl, 0.1 g MgSO4, and 5 g disodium succinate amended with 20 mL steri-filtered 50% (w/v) glucose following autoclaving for 20 min at 121 °C) (9) in a 2 L Fernbach flask. Cultures were grown at 30 °C on an orbital shaker at 180 rpm. Microbial growth was monitored by OD600nm and cultures were harvested when growth reached late log phase with a positive Fe(III)-chrome azurol assay response. (43) Culture supernatants were obtained by centrifugation at 6000 rpm for 30 min at 4 °C (SLA-3000 rotor, Thermo Scientific). To extract Gbt, the culture supernatant was decanted and shaken with 100 g Amberlite® XAD-4 resin. The XAD-4 resin was prepared by washing with methanol and then equilibrating with Milli-Q water. The resin and supernatant were allowed to equilibrate for 4 h at 120 rpm. The resin was filtered from the supernatant and washed with 0.5 L Milli-Q water. The adsorbed organics were eluted with 80% aq. methanol. The eluent was concentrated under vacuum and stored at 4 °C. Gbt was purified from the eluent by semipreparative HPLC on a YMC 20 x 250 mm C18-AQ column, with a linear gradient of 20%–80% methanol (0.05% trifluoroacetic acid) over 40 min, yielding 20 mg from 1 L culture.

Preparation of Isotopically Enriched Gbt

Isotopically enriched Gbt samples were isolated following the same protocols as nonlabeled Gbt. Amberlite® XAD-4 was freshly prepared for each isotopic study. 15N labeling of Gbt was accomplished using 15NH4Cl as the sole nitrogen source. Three amino acids (10 mM l-Arg, 10 mM l-Orn, and 10 mM l-Cit) were tested as possible substrates for conversion to Gra by addition to the 15NH4Cl MM9 medium (500 mL). For the preparation of 13C15N-Gra-enriched Gbt, 1 mM 13C15N-l-Arg was supplemented into MM9 as outlined above.

Photolysis of Apo-Gbt

Photolysis of apo-Gbt was carried out in a 3 mL quartz cuvette with a 75 mm stir bar or in a quartz NMR tube. An Oriel Instruments Hg(Ar) (No. 6035) pen lamp was used as the UV source. Where mentioned, a bandpass filter (Edmund Optics 253.7 nm filter, 25.00 mm diameter, 40.00 nm full width at half maximum) was used to selectively irradiate at 253.7 nm. Samples were dissolved in an aqueous buffer (5 mM MOPS, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, or Na2HPO4) at pH 8.0. For NMR analysis, 99.9% purity D2O was used in place of Milli-Q H2O at pD 8.0.

Photolysis of Fe-Gbt

Fe-Gbt was prepared in Na2HPO4 (25 mM, pH 8) in a 1:1 ratio of apo-Gbt and Fe(III) (FeCl3·6H2O, 2.58 ± 0.04 mM in 25 mM HCl) and was allowed to equilibrate for 20 h. The Fe(III) stock was standardized spectrophotometrically with 1,10 phenanthroline (510 nm, ε 1.1 × 104 M–1 cm–1) and hydroxylamine as a reducing agent. Photolysis of Fe-Gbt was carried out in a quartz cuvette with a 75 mm stir bar. An Oriel Instrument Hg(Ar) (No. 6035) pen lamp was used as the UV source equipped with a bandpass filter (253.7 nm, Edmund Optics).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.2c00593.

  • 1H, 13C, HSQC, 1H–13C HMBC, 1H–15N HMBC, 1H–1H COSY, 13C–13C COSY, 13C INADEQUATE, and TOCSY NMR spectra, chemical shift tables, UPLC-MS spectra for isotopically labeled apo-Gbt and Gbt photoproduct, multiple sequence alignment of GrbD and GrbE, and biosynthesis of l-Arg (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Christina Makris - Department of Chemistry & Biochemistry, University of California, Santa Barbara, California 93106-9510, United StatesOrcidhttps://orcid.org/0000-0001-5836-3195
    • Jeffrey R. Carmichael - Department of Chemistry & Biochemistry, University of California, Santa Barbara, California 93106-9510, United States
    • Hongjun Zhou - Department of Chemistry & Biochemistry, University of California, Santa Barbara, California 93106-9510, United States
  • Author Contributions

    C.M. and J.R.C. contributed equally to this study.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We are grateful for support from the US National Science Foundation, CHE-2108596. We thank R. Behrens for assistance with the mass spectrometer. This work was supported in part by NSF Major Research Instrumentation award, MRI-1920299, for magnetic resonance instrumentation. The research reported here also made use of the shared facilities of the UCSB MRSEC (NSF DMR 172056), a member of the Materials Research Facilities Network (www.mrfn.org).

References

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  • Abstract

    Figure 1

    Figure 1. Selected N–N bonded compounds. C-diazeniumdiolate in alanosine, fragin, and leudiazen are shown in blue, the N-diazeniumdiolate in DETA NONOate is shown in red, and the N-nitrosourea in streptozocin is shown in green.

    Figure 2

    Figure 2. (a) Structure of Gbt with Gra in inset; (b) BGC of Gbt.

    Figure 3

    Figure 3. UV photolysis of apo-Gbt. (a) UV-absorption spectra of apo-Gbt (44 μM) as a function of the time of photolysis (253.7 nm bandpass filter; 0–6 h in 5 mM MOPS pH 8.0). From 0 to 1 h, spectra were obtained at 4 min intervals; from 1 to 2 h, spectra were obtained at 10 min intervals; and from 2 to 3 h, spectra were obtained at 20 min intervals. Inset showing the isosbestic point between time 0 and 1 h; (b) MS of apo-Gbt; (c) MS of the photoproduct of Gbt.

    Figure 4

    Figure 4. Isotope feeding studies demonstrate l-Gra originates from l-Arg. (a) Gbt isolated from the P. graminis DSM 17151 culture grown on 15NH4Cl and 14N-Arg. Isotopic mass of Gbt at m/z 839 is present along with two mass losses of 14NO; (b) Gbt isolated from the P. graminis DSM 17151 culture grown on 14NH4Cl and 13C615N4-Arg. Isotopic mass of Gbt at m/z 851 is present along with two mass losses of 15NO; (c) Scheme depicting isotopic labeling of Gra originating from 14N-Arg or from 13C615N4-Arg.

    Figure 5

    Figure 5. Multiplicity edited 1H–13C HSQC NMR spectra show the formation of E and Z oxime isomers upon photolysis of 13C15N-Gra-enriched-Gbt (pD 8, 6.2 mM, Pi in 99.9% D2O). (a) 1H–13Cδ correlations in Gra residues of apo-Gbt at 4.20, 61.66 ppm and 4.22, 61.93 ppm; (b) region of HSQC showing disappearance of the 1H–13Cδ correlations in the photoproduct; (c) new downfield 1H–13C HSQC correlations in photolyzed 13C15N-Gra-enriched Gbt, with chemical shifts consistent with E/Z oxime isomers; (d) scheme showing release of NO from Gra yields E and Z oxime isomers but not a nitroso photoproduct. Spectra collected in D2O.

    Figure 6

    Figure 6. Photolysis of 13C15N-Gra-enriched-Gbt yields E/Z oxime isomers.

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    • 1H, 13C, HSQC, 1H–13C HMBC, 1H–15N HMBC, 1H–1H COSY, 13C–13C COSY, 13C INADEQUATE, and TOCSY NMR spectra, chemical shift tables, UPLC-MS spectra for isotopically labeled apo-Gbt and Gbt photoproduct, multiple sequence alignment of GrbD and GrbE, and biosynthesis of l-Arg (PDF)


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