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Biosynthesis of a conserved glycolipid anchor for Gram-negative bacterial capsules

Nature Chemical Biology volume 15, pages 632–640 (2019)Cite this article

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Abstract

Several important Gram-negative bacterial pathogens possess surface capsular layers composed of hypervariable long-chain polysaccharides linked via a conserved 3-deoxy-β-d-manno-oct-2-ulosonic acid (β-Kdo) oligosaccharide to a phosphatidylglycerol residue. The pathway for synthesis of the terminal glycolipid was elucidated by determining the structures of reaction intermediates. In Escherichia coli, KpsS transfers a single Kdo residue to phosphatidylglycerol; this primer is extended using a single enzyme (KpsC), possessing two cytidine 5′-monophosphate (CMP)-Kdo-dependent glycosyltransferase catalytic centers with different linkage specificities. The structure of the N-terminal β-(2→4) Kdo transferase from KpsC reveals two α/β domains, supplemented by several helices. The N-terminal Rossmann-like domain, typically responsible for acceptor binding, is severely reduced in size compared with canonical GT-B folds in glycosyltransferases. The similar structure of the C-terminal β-(2→7) Kdo transferase indicates a past gene duplication event. Both Kdo transferases have a narrow active site tunnel, lined with key residues shared with GT99 β-Kdo transferases. This enzyme provides the prototype for the GT107 family.

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Fig. 1: Unrooted phylogenetic tree of KpsS and KpsC homologs from T. dismutans and pathogens of humans and livestock.
Fig. 2: Biochemical characterization of KpsC-NTd/KpsC-CTd and KpsCEc.
Fig. 3: Structural characterization of the in vitro KpsS product.
Fig. 4: KpsC structure analysis.
Fig. 5: E. coli KpsC-N active site organization.
Fig. 6: Model for assembly of CPS in a KpsS- and KpsC-dependent pathway.

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Data availability

Crystallographic datasets have been deposited in the PDB repository under accession codes 6MGB (KpsC-NTd), 6MGC (KpsC-NEc) and 6MGD (KpsC-CTd). Datasets will become publicly available upon manuscript acceptance. Raw NMR and MS data are available from the corresponding authors upon request.

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Acknowledgements

These studies were supported by funding from the Canadian Institutes of Health Research (C.W.), the Natural Sciences and Engineering Research Council (T.L.L., M.S.K. and C.W.) and the Canadian Glycomics Network (C.W.). T.L.L. and C.W. hold Canada Research Chairs and L.D. is a recipient of a Natural Sciences and Engineering Research Council Postgraduate Scholarship. The authors thank W. Wakarchuk for help and advice concerning the HPLC strategy to resolve product chain lengths.

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  1. These authors contributed equally: Liam Doyle, Olga G. Ovchinnikova

Authors and Affiliations

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

    Liam Doyle, Olga G. Ovchinnikova, Katharine Myler, Evan Mallette, Matthew S. Kimber & Chris Whitfield

  2. Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

    Bo-Shun Huang & Todd L. Lowary

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Contributions

L.D. performed the cloning of constructs expressing KpsC, biochemically characterized and crystallized the enzymes and participated in determining and interpreting the structures. O.G.O. performed HPLC experiments and the structural analysis of the KpsS product. K.M. performed in vitro experiments with the NBD-PG lipid acceptor. E.M. collected X-ray data for KpsC and assisted L.D. with crystallographic refinements. B.-S.H. synthesized the synthetic oligosaccharide acceptors for KpsC. T.L.L. oversaw the work of B.-S.H. and contributed to data analysis; M.S.K. assisted with crystallographic refinements, oversaw the work of E.M. and the collection and interpretation of the crystallographic data. C.W. conceived the project and oversaw the biochemical studies and data analysis performed by L.D. and O.G.O. L.D., O.G.O., M.S.K. and C.W. prepared the initial draft of the paper 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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Doyle, L., Ovchinnikova, O.G., Myler, K. et al. Biosynthesis of a conserved glycolipid anchor for Gram-negative bacterial capsules. Nat Chem Biol 15, 632–640 (2019). https://doi.org/10.1038/s41589-019-0276-8

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