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

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.


■ INTRODUCTION
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 Cdiazeniumdiolate 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 Dthreo β-hydroxyAsp residues of which each Gra and βhydroxyaspartic acid (β-OH-Asp) coordinate Fe(III). 12 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 hemedioxygenase-like domain of SznF hydroxylates N ω -methyl-L-Arg at both the guanidinium N δ and the unmethylated N ω positions. 19−21 Following hydroxylation, the cupin domain of SznF is proposed to catalyze the oxidative rearrangement of N δ -hydroxy-N ω -hydroxy-N ω -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 N δ -hydroxylases, 22−24 and DcsA, which is a N ω -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.   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 NO 2 − ( 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 CO 2 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 λ max 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 (λ max 220 nm) compared to pH 8 (λ max 248 nm).
Isotopic Labeling Establishes Gra Originates from Arg. To investigate the origin of Gra, we employed 13 C and 15 N isotopic feeding studies in the growth of P. graminis DSM 17151. When this strain is grown with 15 Figure S10). 31 The 15 Nlabeling in Gly, each Thr and β-OH-Asp must result from 15 NH 3 released from 13 C 15 N-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 15 NH 3 in the biosynthesis of these amino acids. The M + 17 Gbt isotopic mass is consistent with the incorporation of one 15 N-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 15 NH 3 -labeled amino acids. Incorporation of 13 C 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 Lornithine; 17 therefore we also investigated whether 14 Nornithine and 14 N-citrulline added to the P. graminis DSM 17151 growth medium with 15 NH 4 Cl 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 14 N-L-Orn or Photolysis of Isotopically Labeled Gbt Reveals the Photoproduct Is an Oxime. The 13 C 15 N-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% D 2 O), 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 1 H ( 13 C and 15 N decoupled) and 13 C NMR spectra show two new 1 H resonances at 6.90 and 7.53 ppm and two new 13 C resonances at 152.72 and 153.10 ppm not present in Gbt (Figures 5c and S24−S26). The 1 H-13 C HSQC spectrum of the photoproduct shows that the two new 1 H resonances correlate to the two new 13 C resonances ( Figure  5c). The formation of an oxime is consistent with the Cδ of the Gra residues undergoing a change from sp 3 to sp 2 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 1 H and 13 C shifts of both Gra residues in apo-Gbt (Figure 5a) disappear upon photolysis (Figure 5b). Coupling between 15 N, 13 C, and 1 H nuclei on the 13 C 15 N-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 15 N and two 13 C nuclei (Figures S11 and S16). HSQC resonances from the remaining amino acid residues in Gbt derive from the natural abundance of 13 C. In comparison, the 1 H− 13 C correlations of Cα, Cβ, Cγ, and Cδ in 13 C-enriched Gra are apparent, including the disappearance of the 1 H− 13 C 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 Spectrosco-pY (TOCSY) NMR experiment establishes that the E and Z oxime conformations are present in the complete 1 H 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 1 H 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.

■ CONCLUSIONS
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 1 H and 13 C resonances in the 13 C 15 N-enriched Gbt photoproduct is consistent with the formation of an oxime. The 0.6 ppm difference between the new 1 H 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 13 C 15 N-Gbt photoproduct is consistent with release of 15 NO 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 13 C 6 15 N 4 L-Arg establishes that it is the precursor to L-Gra, with all 13 C's and 15 N'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 N δ and N ω 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.
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 ACS Chemical Biology pubs.acs.org/acschemicalbiology Articles 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 Cdiazeniumdiolate 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 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 ( 1 H, 13 13 C decoupling). Spectra were collected in DMSO-d 6 or D 2 O. Spectra were indirectly referenced by the residual solvent peak or 2 H 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 ddH 2 O (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 K 2 HPO 4 , 2 g KH 2 PO 4 , 0.59 g NaCl, 1 g NH 4 Cl, 0.1 g MgSO 4 , 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 OD 600nm 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. 15 N labeling of Gbt was accomplished using 15 NH 4 Cl 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 15 NH 4 Cl MM9 medium (500 mL). For the preparation of 13 C 15 N-Gra-enriched Gbt, 1 mM 13 C 15 N-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)-1piperazineethanesulfonic acid, or Na 2 HPO 4 ) at pH 8.0. For NMR analysis, 99.9% purity D 2 O was used in place of Milli-Q H 2 O at pD 8.0.
Photolysis of Fe-Gbt. Fe-Gbt was prepared in Na 2 HPO 4 (25 mM, pH 8) in a 1:1 ratio of apo-Gbt and Fe(III) (FeCl 3 ·6H 2 O, 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 × 10 4 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).

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.2c00593. 1 H, 13 C, HSQC, 1 H− 13 C HMBC, 1 H− 15 N HMBC, 1 H− 1 H COSY, 13 C− 13 C COSY, 13 C 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) 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).