Natural products with rare functional groups are likely to be constructed by unique biosynthetic enzymes. One such rare functional group is the O-methyl nitronate, which can undergo [3 + 2] cycloaddition reactions with olefins in mild conditions. O-methyl nitronates are found in some natural products; however, how such O-methyl nitronates are assembled biosynthetically is unknown. Here we show that the assembly of the O-methyl nitronate in the natural product enteromycin carboxamide occurs via activation of glycine on a peptidyl carrier protein, followed by reaction with a diiron oxygenase to give a nitronate intermediate and then with a methyltransferase to give an O-methyl nitronate. Guided by the discovery of this pathway, we then identify related cryptic biosynthetic gene cassettes in other bacteria and show that these alternative gene cassettes can, instead, facilitate oxidative denitrification of glycine-derived nitronates. Altogether, our work reveals bifurcating pathways from a central glycine-derived nitronate intermediate in bacteria.
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We thank the Natural Science and Engineering Research Council of Canada, the Canadian Institutes of Health Research and the Michael Smith Foundation for Health Research for financial support. We thank T. Huan and H. Chen for assistance with the mass spectrometry analysis, M. Ezhova for collecting the NMR data, A. Henderson for assistance on the bioinformatics analysis and P. Daniel‐Ivad and J. Hedges for feedback on the manuscript.
The authors declare no competing interests.
Peer review information Nature Chemistry thanks Michelle Chang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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a, HPLC traces of extracts and purified compounds. I) Fermentation of Streptomyces achromogenes var. streptozoticus NRRL 3125, where we purified compounds E-2 and 1 by semi-preparative HPLC. II) Fermentation of Streptomyces achromogenes subsp. streptozoticus NRRL 2697, which is a previously reported producer of enteromycin and 25, although no enteromycin production was observed in these fermentation conditions in our laboratory. III) Purified Peak I (assigned as Z-2) analysed after several hours standing at room temperature, when some has converted to Peak II (assigned as E-2). IV) Purified E-2 run on HPLC after one week standing at RT, with minimal conversion to Peak I (assigned as Z-2). V) Purified Peak III (assigned as 1) analysed after 3 d standing at RT. b, Specific UV absorbance of peaks I, II, and III.
Trace I, NRRL 3125 wild-type; Trace II, ΔetmG inactivation mutant; Trace III, ΔetmB inactivation mutant; Trace IV, ΔetmE inactivation mutant. Production of 1 and 2 is fully abolished in all three mutants.
a, Scheme of degradation and derivatization of formaldehyde with DNPH. b, EIC from LC-MS analysis of derivatized mixtures. m/z 198 and 210 were extracted, corresponding to DNPH and 10, respectively. I) Formaldehyde standard; II) Control reaction without EtmE; III) EtmE reaction. Negative mode was used for MS detection. 10 was detected in EtmE reaction, which confirmed the formation of O-methylnitronate in EtmE reaction.
Extended Data Fig. 5 Putative late steps in biosynthetic pathway of enteromycin-carboxamide (1) and U-15774 (2).
a, Biosynthetic gene cluster of 1. b, Putative late steps of the biosynthetic pathway. We propose that condensation of Gly-derived O-methyl nitronate 9 and Asp-derived β-aminopropionate 5, catalysed by putative 3-oxoacyl-[acyl-carrier-protein] synthase EtmF, gives a dipeptide intermediate, which is further modified via dehydrogenation (by putative dehydrogenases EtmK or EtmL), thioester hydrolysis (by putative thioesterase EtmQ) and transamination (by putative asparagine synthetase EtmT) to form 1. Remarkably, dehydrogenation may occur on 5 before condensation, as displayed in Fig. 1d of main text. A very recent study on the biosynthetic pathway to bolagladins shows that an enzyme related to EtmL, called BolQ, desaturates a β-aminopropionate unit, suggesting EtmL could have a similar function in 1 biosynthesis51,52. Additional genes in the enteromycin gene cluster include etmC and etmM, which encode putative 4'- phosphopantetheinyl transferases, which we propose activate two peptidyl carrier proteins EtmD and EtmI, respectively. The gene etmN encodes a putative S-adenosylmethionine synthetase, which might catalyse the formation of SAM for SAM-dependent methyltransferase EtmE. For the biosynthesis of oxime 2, one scenario is that it derives from a nitroso intermediate 7, detected in the EtmA reaction, which reacts with 5 directly catalysed by EtmF. However, the lack of 2 from ΔetmE (Extended Data Fig. 3) conflicts with this scenario. Previous studies revealed that 1 can convert to 2 under heating. We observed that purified 1 decomposed slowly to E- and Z-2 even at room temperature (Extended Data Fig. 2). Those results suggest a possible alternate nonenzymatic pathway to generate 2 from 1 in vivo.
a, Scheme of enzymatic reactions and chemical derivatization using DNPH. The nitroacetyl-S-carrier protein was generated from EtmA or UnkA reactions with unlabeled or labeled Gly as the substrate, as described for each panel. b,c, LC-MS analysis of derivatized mixtures in negative ion mode. b, Extracted-ion chromatogram (EIC) from LC-MS analysis, with m/z 198, 253 and 254 extracted, corresponding to DNPH, 15 and 15’, respectively. I) Derivatization of a glyoxylate chemical standard; II) Derivatization of the reaction with unlabeled Gly as the substrate; III) Derivatization of the reaction with 2-13C-15N-Gly as the substrate. c, MS signals displayed by each peak. I) Derivatization of the reaction with unlabeled Gly as the substrate; II) Derivatization of the reaction with 2-13C-15N-Gly as the substrate. Two peaks, consistent with DNPH-derivatized glyoxylate 15, were observed, which are assigned as isomers of carbon-nitrogen double bond (E-/Z-15), although which peak is E- or Z-form is unknown. As expected, comparing with the results of unlabeled-Gly added mixture, we detected +1 Da peak in the mixture of 2-13C-15N-Gly added, which is consistent with loss of one nitrogen and confirmed the formation of nitronate.
A sequence-similarity network was generated using the EFI-Enzyme Similarity Tool, which analyses all proteins in UniProt53. Edges connect sequences that align with an E-value threshold of 1.0 x 10−36. Sequences with identities exceeding 90 % are represented by a single node. The network was visualized by Cytoscape54 using prefuse force directed openCL layout. Four identified clusters labeled are listed that contain known biosynthetic enzymes AurF17,18, SpnF55, CmlI19, ObiL (ObaC)32,56, AzoC33, BezJ57, ActI58, MatA59, TsnB734, HamC38, PvfB36,39 and AlmD35, all of them belonging to Pfam 11583.
The schemes show characterized reactions of members of Pfam 11583. Not shown are two CmlI and ObiL (ObaC), which catalyse similar reactions to AurF in in the biosynthesis of chloramphenicol19 and obafluorin32,56, respectively. Note that ActI and MatA have not yet been experimentally characterized, and putative reactions are shown58,59.
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He, HY., Ryan, K.S. Glycine-derived nitronates bifurcate to O-methylation or denitrification in bacteria. Nat. Chem. 13, 599–606 (2021). https://doi.org/10.1038/s41557-021-00656-8