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A bifunctional O-antigen polymerase structure reveals a new glycosyltransferase family

Nature Chemical Biology volume 16, pages 450–457 (2020)Cite this article

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Abstract

Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→ 3)-α-Galp-(1 → 3)-β-Galf-(1 →). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-Å resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans.

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Fig. 1: Structures and biosynthesis of OPSs based on the O2a antigen.
Fig. 2: Identification of two GT modules in WbbM.
Fig. 3: Overall structure of WbbM.
Fig. 4: Details of the WbbM catalytic sites.
Fig. 5: Model for the membrane-tethered WbbM trimer.

Data availability

The crystallographic dataset for native WbbM has been deposited in the PDB repository under accession code 6U4B. Raw NMR and mass spectrometry data are available from the corresponding authors upon request.

Code availability

Custom Python scripts used in this study are available at https://github.com/bclarke2/wbbM_glf_scripts.

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Acknowledgements

The authors wish to thank S. Labiuk at the Canadian Light Source for help with data collection and S. Al-Abdul-Wahid and A. Lo for technical support with NMR spectroscopy. These studies were supported by individual Natural Sciences and Engineering Research Council Discovery Grants awarded to M.S.K., T.L.L., and C.W. T.L.L. and C.W. hold Canada Research Chairs and S.D.K. is a recipient of a Natural Sciences and Engineering Research Council Postgraduate Scholarship.

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada

    Bradley R. Clarke, Olga G. Ovchinnikova, Evelyn R. Kamski-Hennekam, Russel Gitalis, Evan Mallette, Steven D. Kelly, Matthew S. Kimber & Chris Whitfield

  2. Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

    Ryan P. Sweeney & Todd L. Lowary

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Contributions

B.R.C. performed all of the biochemical experiments and phylogenetics analysis shown. O.G.O. performed NMR experiments and participated in interpretation of mass spectrometry data. R.P.S. synthesized the synthetic oligosaccharide acceptors. E.R.K.-H. generated site-directed mutants and performed initial activity assessments. R.G., E.R.K.-H. and E.M. crystallized WbbM and collected X-ray data. S.D.K. purified LPS samples, performed mass spectrometry analysis, together with O.G.O. T.L.L. oversaw the work of R.P.S. and contributed to data analysis. M.S.K. oversaw the work of E.R.K.-H., R.G. and E.M. assisted with crystallographic refinements, identified targets for mutagenesis and performed bioinformatic analysis of the membrane-associated C terminus. C.W. conceived the project and oversaw the biochemical studies and data analysis performed by B.R.C., O.G.O. and S.D.K. The initial draft of the paper was prepared by B.R.C., O.G.O., M.S.K. and C.W., and all authors made contributions to the final version.

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Correspondence to Matthew S. Kimber or Chris Whitfield.

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Clarke, B.R., Ovchinnikova, O.G., Sweeney, R.P. et al. A bifunctional O-antigen polymerase structure reveals a new glycosyltransferase family. Nat Chem Biol 16, 450–457 (2020). https://doi.org/10.1038/s41589-020-0494-0

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