Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Nature Catalysis volume 1, pages 977–984 (2018)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

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.

References

  1. Batelaan, J. G., Barnick, J. W. F. K., van der Baan, J. L. & Bickelhaupt, F. The structure of the antibiotic K16. I. The dipeptide side chain. Tetrahedron Lett. 13, 3103–3106 (1972).

    Article  Google Scholar 

  2. Batelaan, J. G., Barnick, J. W. F. K., van der Baan, J. L. & Bickelhaupt, F. The structure of the antibiotic K16. II. Chromophore and total structure. Tetrahedron Lett. 13, 3107–3110 (1972).

    Article  Google Scholar 

  3. Schipper, D., van der Baan, J. L. & Bickelhaupt, F. Biosynthesis of malonomicin. Part 1. 13C nuclear magnetic resonance spectrum and feeding experiments with 13C-labelled precursors. J. Chem. Soc. Perkin Trans. 10, 2017–2022 (1979).

    Article  Google Scholar 

  4. Chan, Y. A., Podevels, A. M., Kevany, B. M. & Thomas, M. G. Biosynthesis of polyketide synthase extender units. Nat. Prod. Rep. 26, 90–114 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Stenflo, J., Fernlund, P., Egan, W. & Roepstorff, P. Vitamin K dependent modifications of glutamic acid residues in prothrombin. Proc. Natl Acad. Sci. USA 71, 2730–2733 (1974).

    Article  CAS  PubMed  Google Scholar 

  6. Esmon, C. T., Sadowski, J. A. & Suttie, J. W. A new carboxylation reaction. J. Biol. Chem. 250, 4744–4748 (1975).

    CAS  PubMed  Google Scholar 

  7. Wu, S.-M., Morris, D. P. & Stafford, D. W. Identification and purification to near homogeneity of the vitamin K-dependent carboxylase. Proc. Natl Acad. Sci. USA 88, 2236–2240 (1991).

    Article  CAS  PubMed  Google Scholar 

  8. Wu, S.-M., Cheung, W.-F., Frazier, D. & Stafford, D. W. Cloning and expression of the cDNA for human gamma-glutamyl carboxylase. Science 254, 1634–1636 (1991).

    Article  CAS  PubMed  Google Scholar 

  9. Dowd, P., Ham, S. W. & Geib, S. J. Mechanism of action of vitamin K. J. Am. Chem. Soc. 113, 7734–7743 (1991).

    Article  CAS  Google Scholar 

  10. Dowd, P., Ham, S. W. & Hershline, R. Role of oxygen in the vitamin K-dependent carboxylation reaction: incorporation of a second atom of 18O from molecular oxygen-18O2 into vitamin K oxide during carboxylase activity. J. Am. Chem. Soc. 114, 7613–7617 (1992).

    Article  CAS  Google Scholar 

  11. Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. Vitamin K and energy transduction: a base strength amplification mechanism. Science 269, 1684–1691 (1995).

    Article  CAS  PubMed  Google Scholar 

  12. Fasco, M. J., Preusch, P. C., Hildebrandt, E. & Suttie, J. W. Formation of hydroxyvitamin K by vitamin K epoxide reductase of warfarin-resistant rats. J. Biol. Chem. 258, 4372–4380 (1983).

    CAS  PubMed  Google Scholar 

  13. Bell, R. G. & Matschiner, J. T. Warfarin and the inhibition of vitamin K activity by an oxide metabolite. Nature 237, 32–33 (1972).

    Article  CAS  PubMed  Google Scholar 

  14. Rishavy, M. A. et al. The vitamin K-dependent carboxylase has been acquired by Leptospira pathogens and shows altered activity that suggests a role other than protein carboxylation. J. Biol. Chem. 280, 34870–34877 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Kobylarz, M. J. et al. Synthesis of l-2,3-diaminopropionic acid, a siderophore and antibiotic precursor. Chem. Biol. 21, 379–388 (2014).

    Article  CAS  PubMed  Google Scholar 

  16. Rishavy, M. A. et al. Brønsted analysis reveals Lys218 as the carboxylase active site base that deprotonates vitamin K hydroquinone to initiate vitamin K-dependent protein carboxylation. Biochemistry 45, 13239–13248 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Rishavy, M. A. & Berkner, K. L. Insight into the coupling mechanism of the vitamin K-dependent carboxylase: mutation of histidine 160 disrupts glutamic acid carbanion formation and efficient coupling of vitamin K epoxidation to glutamic acid carboxylation. Biochemistry 47, 9836–9846 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Alderson, G., Goodfellow, M. & Minnikin, D. E. Menaquinone composition in the classification of Streptomyces and other sporoactinomycetes. Microbiology 131, 1671–1679 (1985).

    Article  CAS  Google Scholar 

  19. Hiratsuka, T. et al. An alternative menaquinone biosynthetic pathway operating in microorganisms. Science 321, 1670–1673 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Nowicka, B. & Kruk, J. Occurrence, biosynthesis and function of isoprenoid quinones. Biochim. Biophys. Acta 1797, 1587–1605 (2010).

    Article  CAS  PubMed  Google Scholar 

  21. Schultz, J. HTTM, a horizontally transferred transmembrane domain. Trends Biochem. Sci. 29, 4–7 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Cobb, R. E., Wang, Y. & Zhao, H. High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth. Biol. 4, 723–728 (2015).

    Article  CAS  PubMed  Google Scholar 

  23. Davidsen, J. M., Bartley, D. M. & Townsend, C. A. Nonribosomal propeptide precursor in nocardicin A biosynthesis predicted from adenylation domain specificity dependent on the MbtH family protein NocI. J. Am. Chem. Soc. 135, 1749–1759 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Reimer, D., Pos, K. M., Thines, M., Grün, P. & Bode, H. B. A natural prodrug activation mechanism in nonribosomal peptide synthesis. Nat. Chem. Biol. 7, 888–890 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Luo, Y. et al. Validation of the intact zwittermicin A biosynthetic gene cluster and discovery of a complementary resistance mechanism in Bacillus thuringiensis. Antimicrob. Agents Chemother. 55, 4161–4169 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Marahiel, M. A., Stachelhaus, T. & Mootz, H. D. Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem. Rev. 97, 2651–2673 (1997).

    Article  CAS  PubMed  Google Scholar 

  27. Bergendahl, V., Linne, U. & Marahiel, M. A. Mutational analysis of the C-domain in nonribosomal peptide synthesis. Eur. J. Biochem. 269, 620–629 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Webb, M. R. A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc. Natl Acad. Sci. USA 89, 4884–4887 (1992).

    Article  CAS  PubMed  Google Scholar 

  29. Pavela-Vrancic, M., Dieckmann, R. & Von Döhren, H. ATPase activity of non-ribosomal peptide synthetases. Biochim. Biophys. Acta 1696, 83–91 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Stachelhaus, T., Mootz, H. D. & Marahiel, M. A. The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem. Biol. 6, 493–505 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Szókán, G., Mezö, G. & Hudecz, F. Application of Marfey’s reagent in racemization studies of amino acids and peptides. J. Chromatogr. A 444, 115–122 (1988).

    Article  Google Scholar 

  32. Bochmann, S. M., Spieß, T., Kötter, P. & Entian, K. D. Synthesis and succinylation of subtilin-like lantibiotics are strongly influenced by glucose and transition state regulator AbrB. Appl. Environ. Microbiol. 81, 614–622 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Cochrane, S. A., Surgenor, R. R., Khey, K. M. W. & Vederas, J. C. Total synthesis and stereochemical assignment of the antimicrobial lipopeptide cerexin A1. Org. Lett. 17, 5428–5431 (2015).

    Article  CAS  PubMed  Google Scholar 

  34. Spieß, T., Korn, S. M., Kötter, P. & Entian, K. D. Activation of histidine kinase SpaK is mediated by the N-terminal portion of subtilin-like lantibiotics and is independent of lipid II. Appl. Environ. Microbiol. 81, 5335–5343 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Chan, W. C., Bycroft, B. W., Leyland, M. L., Lian, L.-Y. & Roberts, G. C. K. A novel post-translational modification of the peptide antibiotic subtilin: isolation and characterization of a natural variant from Bacillus subtilis A.T.C.C. 6633. Biochem. J. 291, 23–27 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Rutherford, K. et al. Artemis: sequence visualization and annotation. Bioinformatics 16, 944–945 (2000).

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

Author notes

  1. These authors contributed equally: Brian J. C. Law, Ying Zhuo.

Authors and Affiliations

  1. School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK

    Brian J. C. Law, Ying Zhuo, Michael Winn, Daniel Francis, Yingxin Zhang, Lujing Ren & Jason Micklefield

  2. Department of Biochemistry, University of Cambridge, Cambridge, UK

    Markiyan Samborskyy, Annabel Murphy & Peter F. Leadlay

Authors
  1. Brian J. C. Law
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ying Zhuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael Winn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel Francis
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yingxin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Markiyan Samborskyy
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Annabel Murphy
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lujing Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Peter F. Leadlay
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jason Micklefield
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Jason Micklefield.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

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

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41929-018-0178-2

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing