title: Biochemical Characterization of Bifunctional 3-Deoxy-β-d-manno-oct-2-ulosonic Acid (β-Kdo) Transferase KpsC from Escherichia coli Involved in Capsule Biosynthesis.
abstract: 3-Deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an essential component of bacterial lipopolysaccharides, where it provides the linkage between lipid and carbohydrate moieties. In all known LPS structures, Kdo residues possess α-anomeric configurations, and the corresponding inverting α-Kdo transferases are well characterized. Recently, it has been shown that a large group of capsular polysaccharides from Gram-negative bacteria, produced by ATP-binding cassette transporter-dependent pathways, are also attached to a lipid anchor through a conserved Kdo oligosaccharide. In the study reported here, the structure of this Kdo linker was determined by NMR spectroscopy, revealing alternating β-(2→4)- and β-(2→7)-linked Kdo residues. KpsC contains two retaining β-Kdo glycosyltransferase domains belonging to family GT99 that are responsible for polymerizing the β-Kdo linker on its glycolipid acceptor. Full-length Escherichia coli KpsC was expressed and purified, together with the isolated N-terminal domain and a mutant protein (KpsC D160A) containing a catalytically inactivated N-terminal domain. The Kdo transferase activities of these proteins were determined in vitro using synthetic acceptors, and the reaction products were characterized using TLC, mass spectrometry, and NMR spectroscopy. The N- and C-terminal domains were found to catalyze formation of β-(2→4) and β-(2→7) linkages, respectively. Based on phylogenetic analyses, we propose the linkage specificities of the glycosyltransferase domains are conserved in KpsC homologs from other bacterial species.
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1 and.
authors.forename: Olga G
authors.initials: OG
authors.lastname: Ovchinnikova
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1 and.
authors.forename: Liam
authors.initials: L
authors.lastname: Doyle
authors.affiliation: the Department of Chemistry and Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Bo-Shun
authors.initials: BS
authors.lastname: Huang
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1 and.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Kimber
authors.affiliation: the Department of Chemistry and Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Todd L
authors.initials: TL
authors.lastname: Lowary
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1 and cwhitfie@uoguelph.ca.
authors.forename: Chris
authors.identifier: http://orcid.org/0000-0002-5267-7027
authors.initials: C
authors.lastname: Whitfield
mesh_terms: D016667:Bacterial Capsules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008070:Lipopolysaccharides / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D000072417:Protein Domains; D013400:Sugar Acids / Q000737:chemistry / Q000378:metabolism; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D009844:Oligosaccharides; D013400:Sugar Acids; D014166:Transferases
keywords: Escherichia coli (E. coli); Kdo; capsular polysaccharide; cell surface; glycoconjugate; glycolipid structure; glycosyltransferase; gram-negative bacteria; oligosaccharide