Abstract
Triacsins are an intriguing class of specialized metabolites possessing a conserved N-hydroxytriazene moiety not found in any other known natural products. Triacsins are notable as potent acyl-CoA synthetase inhibitors in lipid metabolism, yet their biosynthesis has remained elusive. Through extensive mutagenesis and biochemical studies, we here report all enzymes required to construct and install the N-hydroxytriazene pharmacophore of triacsins. Two distinct ATP-dependent enzymes were revealed to catalyze the two consecutive N–N bond formation reactions, including a glycine-utilizing, hydrazine-forming enzyme (Tri28) and a nitrite-utilizing, N-nitrosating enzyme (Tri17). This study paves the way for future mechanistic interrogation and biocatalytic application of enzymes for N–N bond formation.
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Data availability
The authors declare that all data supporting the findings of this study are available within the paper, Supplementary information and the Extended data, and/or from the corresponding author on reasonable request.
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Acknowledgements
We thank T.-Y. Huang and B. McCloskey at UC Berkeley for their assistance with initial GC–MS experiments and consultation in designing experiments. We thank M. Zhang from the UC Berkeley Catalysis Center for training with the GC–MS apparatus and aiding with data interpretation, J. Pelton for helping with NMR spectroscopic analysis and W. Yang for helping with ICP–OES analysis. The GC–MS work was made possible by the Catalysis Facility of Lawrence Berkeley National Laboratory, supported by the Director, Office of Science, of the US Department of Energy (contract no. DE-AC02-05CH11231). This research was financially supported by grants to W.Z. from the NIH (nos. R01GM136758 and DP2AT009148) and the Chan Zuckerberg Biohub Investigator Program. We thank Z. Hu for helpful discussions regarding the complex biosynthesis of triacsins. Lastly, we thank Y. Shen for assistance in protein purification and kinetic assays.
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A.D.R.F. designed the experiments, performed all in vitro experiments with purified enzymes, analyzed the data and wrote the manuscript. F.F.T. designed experiments to detect the biosynthetic intermediate, constructed plasmids for protein expression in E. coli and analyzed data. Y.D. analyzed all NMR data. W.C. performed GC–MS experiments and helped analyze NMR data. D.Q.A. helped purify the biosynthetic intermediate. M.S. isolated triacsin A. M.J.D., M.N. and J.G. aided in protein purification, construction of plasmids and repeating biochemical assays for this study. N.A.Z. performed comparative metabolomics work. R.Z. performed FPLC purification for Tri22, Tri28 and individual domains. W.Z. designed the experiments, analyzed the data and wrote the manuscript.
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Peer review information Nature Chemical Biology thanks Jonathan Caranto, Yohei Katsuyama and Jurgen Rohr for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 The decarboxylation event in triacsin biosynthesis.
a) Feeding of unlabeled and labeled glycine to the triacsin producing cultures. HRMS analysis of 3 from a S. aureofaciens culture provided with 10 mM 1-13C-glycine demonstrated that the carboxylate carbon of glycine was retained in triacsin C (3). b) Detection of CO2 production from the Tri109 biochemical assay. GC-MS chromatograms extracted with m/z = 44, demonstrating CO2 production from a Tri109 assay containing 5 as compared with an authentic standard. A 10-ppm mass error tolerance was used for each trace. At least two independent replicates were performed for each assay, and representative results are shown.
Extended Data Fig. 2 In vitro reconstitution of Tri26 and Tri28.
a) EICs showing the production of 7 in an assay containing Tri26, lysine, and NADPH. Tri26 selectively hydroxylates lysine and not ornithine. The calculated mass for 7: m/z = 163.1078 ([M + H]+) is used for each trace, except for the ornithine assay in which the calculated mass for N-OH-Orn was used (m/z = 149.0921 ([M + H]+)). b) EICs demonstrating that the production of 8 is dependent on Tri28, glycine, lysine, and ATP, along with the Tri26 assay components. The calculated mass for 8: m/z = 220.1292 ([M + H]+) is used for each trace. A 10-ppm mass error tolerance was used for each trace. At least three independent replicates were performed for each assay, and representative results are shown.
Extended Data Fig. 3 Characterization of the Tri22-catalyzed reaction product, 11.
a) Schematic of a biochemical assay performed containing HAA, succinyl-CoA, Tri29-31, and Tri22 that is subjected to base hydrolysis, followed by OPTA derivatization to yield 2-HYAA-D. b) EICs showing the generation of 2-HYAA-D from the biochemical assay previously described in comparison with a derivatized 2-HYAA synthesized standard. Omission of Tri22, HAA, or succinyl-CoA from the couple enzymatic reaction resulted in abolishment of 2-HYAA-D. The calculated mass for 2-HYAA-D: m/z = 265.0642 ([M + H]+) is used for each trace. A 10-ppm mass error tolerance was used for each trace. At least three independent replicates were performed for each assay, and representative results are shown.
Extended Data Fig. 4 In vitro analysis of Tri14, a putative thioesterase.
a) EICs demonstrating hydrolysis of lauroyl-S-Tri20 catalyzed by Tri14, resulting in production of lauric acid when compared to an authentic standard. Tri14 was shown to be Tri20 specific and imparted no activity on lauroyl-S-Tri30. The calculated mass for lauric acid: m/z = 199.1701 ([M-H]−) is used for each trace. b) EIC demonstrating inability of Tri14 to hydrolyze hexanoyl-S-Tri20, thus suggesting that Tri14 activity is chain length specific. The calculated mass for hexanoic acid: m/z = 115.0764 ([M-H]−) is used for each trace. A 10-ppm mass error tolerance was used for each trace. At least three independent replicates were performed for each assay, and representative results are shown.
Extended Data Fig. 5 AMP Formation from Tri17 assay and proposed reaction mechanism.
LC-UV detection of AMP at 260 nm from an assay containing 15, nitrite, ATP, and Tri17. Controls lacking enzyme or nitrite result in undetectable amounts of AMP, while the production of AMP was slightly increased in the presence of 15. ATP is proposed to activate nitrite to form a nitrite-AMP intermediate that undergoes a subsequent nucleophilic attack by 15 and tautomerization to yield 1. At least three independent replicates were performed for each assay, and representative results are shown.
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Supplementary Tables 1–3, Figs. 1–16 and Notes 1–5.
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Del Rio Flores, A., Twigg, F.F., Du, Y. et al. Biosynthesis of triacsin featuring an N-hydroxytriazene pharmacophore. Nat Chem Biol 17, 1305–1313 (2021). https://doi.org/10.1038/s41589-021-00895-3
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DOI: https://doi.org/10.1038/s41589-021-00895-3
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