A vitamin K-dependent carboxylase orthologue is involved in antibiotic biosynthesis

Abstract

Vitamin K-dependent carboxylase (VKDC) enzymes modify glutamate residues in mammalian vitamin K-dependent proteins, generating γ-carboxyglutamic acids with malonate moieties that mediate important physiological responses such as blood coagulation. Proteins with sequence similarity to mammalian VKDC are also found in bacteria; however, their function remains unknown. The antibiotic malonomycin from Streptomyces rimosus contains an unusual malonate group, of unknown origin, that is essential for its biological activity. Here, we show that a bacterial VKDC orthologue (MloH) is responsible for the malonic acid moiety in malonomycin. Using CRISPR/Cas9 gene editing, complementation and mutagenesis experiments, this VKDC-like enzyme was shown to α-carboxylate an aspartyl residue within a hybrid polyketide–nonribosomal peptide intermediate during malonomycin biosynthesis. This study reveals a highly unusual biosynthetic pathway to malonic acid-containing metabolites, providing a functional role for VKDC-like proteins in prokaryotes and a vitamin K-dependent carboxylation reaction with a non-proteinogenic substrate.

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Fig. 1: Biosynthesis of malonic acid derivatives.
Fig. 2: Malonomycin (mlo) gene cluster and biosynthetic pathway.
Fig. 3: Substrate selectivity of MloI A2T2 didomain and l-Asp substrate side-chain activation.
Fig. 4: Characterization of compounds from wild-type and engineered strains.

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request. Nucleotide sequences for the malonomycin BGCs are deposited in GenBank (accession numbers MH104948 (S. rimosus paramyceticus R2374) and MH104947 (S. rimosus paromomycinus NRRL 2455). The whole genome shotgun sequence for N. gamkensis NBRC 108242, used for the identification of the malonomycin-like cluster from this strain, was obtained from GenBank National Center for Biotechnology Information reference sequence NZ_BDBM01000044.1. Details on nucleotide sequences and proposed annotations are detailed in the Supplementary Information.

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Acknowledgements

We thank BBSRC (grant BB/K002341/1) and Syngenta for funding. The SYNBIOCHEM Centre (grant BB/M017702/1) and Michael Barber Centre for Mass Spectrometry at the University of Manchester provided access to mass spectrometry instrumentation. J. Vincent and N. Mulholland (Syngenta) are also acknowledged for helpful discussion.

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B.J.C.L., Y. Zhuo, M.W. and J.M. designed the experiments. B.J.C.L., Y.Z., M.W., D.F., Y. Zhang, A.M. and L.R. carried out the experiments. M.S. and P.F.L. sequenced and annotated the genomes. B.J.C.L. and J.M. wrote the manuscript. All authors analysed the data and reviewed the manuscript.

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Correspondence to Jason Micklefield.

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Supplementary Methods, Supplementary Figs 1–16, Supplementary Tables 1–5, Supplementary References

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Law, B.J.C., Zhuo, Y., Winn, M. et al. A vitamin K-dependent carboxylase orthologue is involved in antibiotic biosynthesis. Nat Catal 1, 977–984 (2018). https://doi.org/10.1038/s41929-018-0178-2

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