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Pyridoxal-5′-phosphate-dependent alkyl transfer in nucleoside antibiotic biosynthesis

Nature Chemical Biology volume 16, pages 904–911 (2020)Cite this article

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Abstract

Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5′S,6′S)-5′-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR–GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-l-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5′-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.

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Fig. 1: Structures of representative TL1 inhibitors that contain an N-alkylated ADR–GlyU disaccharide core.
Fig. 2: Spectroscopic analysis of feeding experiments.
Fig. 3: Functional assignment of Mur24.
Fig. 4: Isotopic incorporation from l-Met.
Fig. 5: Functional assignment of Mur23.
Fig. 6: Putative γ-elimination and γ-addition mechanism for the PLP-dependent AdoMet:9 ABTases.

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Data availability

All plasmids and raw data are available upon request. Sequences are deposited at National Center for Biotechnology Information under accession nos.: Mur24, ADZ45336; Mur23, ADZ45335; LipJ, BAJ05886; and SphL, BAO20191.

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Acknowledgements

This work was supported in part by the National Institutes of Health (NIH, grant nos. AI087849 and GM115261), the National Center for Advancing Translational Sciences (grant nos. UL1TR000117 and UL1TR001998), the Deutsche Forschungsgemeinschaft (grant no. DU1095/5-1), the state of Lower Saxony (Lichtenberg doctoral fellowship (CaSuS program) for A.L.), the Konrad-Adenauer-Stiftung (doctoral fellowship for D.W.) and the Fonds der Chemischen Industrie (doctoral fellowship for G.N.). NMR (600 MHz) data were collected at the NMR facility of the Center for Environmental Systems Biochemistry supported in part by the NIH (grant no. DK097215). We thank the Department of Pharmaceutical Sciences and the College of Pharmacy for financial support for MS and NMR instrumentation.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY, USA

    Zheng Cui, Jonathan Overbay, Xiaodong Liu, Minakshi Bhardwaj, Jon S. Thorson & Steven G. Van Lanen

  2. Center for Pharmaceutical Research and Innovation, College of Pharmacy, University of Kentucky, Lexington, KY, USA

    Xiachang Wang, Yinan Zhang & Jon S. Thorson

  3. Jiangsu Key Laboratory for Functional Substance of Chinese Medicine, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, People’s Republic of China

    Xiachang Wang & Yinan Zhang

  4. Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbrücken, Germany

    Anke Lemke, Daniel Wiegmann, Giuliana Niro & Christian Ducho

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Contributions

Z.C. and J.O. performed and analyzed the data for cloning, heterologous expression, and protein functional assignments and biochemical characterization. Z.C., X.W. and M.B. performed MS and NMR spectroscopy of enzymatic products and analyzed the respective data. X.L, Y.Z., A.L., D.W. and G.N. performed the chemical synthesis and analyzed the respective data. Z.C., J.S.T., C.D. and S.G.v.L. conceived the study, directed and planned the research, analyzed the data and wrote the article.

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Correspondence to Steven G. Van Lanen.

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Cui, Z., Overbay, J., Wang, X. et al. Pyridoxal-5′-phosphate-dependent alkyl transfer in nucleoside antibiotic biosynthesis. Nat Chem Biol 16, 904–911 (2020). https://doi.org/10.1038/s41589-020-0548-3

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