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Biochemical Characterization of Bifunctional 3-Deoxy-?-d-manno-oct-2-ulosonic Acid (?-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 ?-anomeric configurations, and the corresponding inverting ?-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 ?-(2?4)- and ?-(2?7)-linked Kdo residues. KpsC contains two retaining ?-Kdo glycosyltransferase domains belonging to family GT99 that are responsible for polymerizing the ?-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 ?-(2?4) and ?-(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.

SUBMITTER: Ovchinnikova OG 

PROVIDER: S-EPMC5076823 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Biochemical Characterization of Bifunctional 3-Deoxy-β-d-manno-oct-2-ulosonic Acid (β-Kdo) Transferase KpsC from Escherichia coli Involved in Capsule Biosynthesis.

Ovchinnikova Olga G OG   Doyle Liam L   Huang Bo-Shun BS   Kimber Matthew S MS   Lowary Todd L TL   Whitfield Chris C  

The Journal of biological chemistry 20160817 41


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 al  ...[more]

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