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

Abstract

Several important Gram-negative bacterial pathogens possess surface capsular layers composed of hypervariable long-chain polysaccharides linked via a conserved -Kdo oligosaccharide to a phosphatidylglycerol residue. The pathway for synthesis of the terminal glycolipid was elucidated by determining 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 CMP-Kdodependent glycosyltransferase catalytic centres with different linkage specificities. The structure of the Nterminal -(2→4)-Kdo transferase from KpsC reveals two α/β domains, supplemented by several additional helices. The N-terminal Rossmann-like domain, typically responsible for acceptor binding, is severely reduced in size compared to canonical GT-B folds in glycosyltransferases. The similar structure of the Cterminal -(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 -Kdo transferases. This enzyme provides the prototype for the GT107 family. repeat-unit glycan, before the completed molecule enters the export pathway via the assembly strategy-defining ATP-binding cassette (ABC) transporter. Activities of KpsC and its individual GT modules were established using synthetic Kdo mono-and disaccharide acceptors attached to aromatic tags 12 . However, the precise structure of the natural acceptor (KpsS product) has not been determined and its reactivity with KpsC GT modules remains unknown.KpsC is a potential target for small-molecule therapeutics development due to its pivotal role in assembling an essential virulence determinant in high-profile encapsulated pathogens, and the absence of β-Kdo in mammals and in many bacterial commensals. Understanding the structure and function of KpsC is an important step in this strategy. The objectives of this study were to establish a definitive structure for the natural glycolipid acceptor for CPS polymerization and elucidate the sequence of addition of Kdo residues in its biogenesis. Here, we also characterize single GT module KpsC homologs from the Gram-negative thermophile, Thermosulfurimonas dismutans. This provided an essential resource to solve enzyme structures from both E. coli and T. dismutans, identifying in a new GT family. ResultsBioinformatic and functional investigation of KpsC. Functional characterization of the E. coli KpsC enzyme is challenging, due to the instability of full-length protein and most truncated derivatives 11,12 . This led us to investigate KpsC homologs, with an emphasis on thermophiles anticipating that these would have improved stability. Using BLAST, we identified candidates in the Gram-negative thermophile, T. dismutans (Supplementary Fig. 1a,b). While most KpsC proteins have dual GT modules, T. dismutans produces two smaller proteins (318 and 348 amino acid residues). Sequence similarity indicates these are homologs of the individual GT modul...

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