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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]

PMID: 27535220

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Project description:Metabolic labeling paired with click chemistry is a powerful approach for selectively imaging the surfaces of diverse bacteria. Herein, we explored the feasibility of labeling the lipopolysaccharide (LPS) of Myxococcus xanthus-a Gram-negative predatory social bacterium known to display complex outer membrane (OM) dynamics-via growth in the presence of distinct azido (-N3) analogues of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Determination of the LPS carbohydrate structure from strain DZ2 revealed the presence of one Kdo sugar in the core oligosaccharide, modified with phosphoethanolamine. The production of 8-azido-8-deoxy-Kdo (8-N3-Kdo) was then greatly improved over previous reports via optimization of the synthesis of its 5-azido-5-deoxy-d-arabinose precursor to yield gram amounts. The novel analogue 7-azido-7-deoxy-Kdo (7-N3-Kdo) was also synthesized, with both analogues capable of undergoing in vitro strain-promoted azide-alkyne cycloaddition (SPAAC) "click" chemistry reactions. Slower and faster growth of M. xanthus was displayed in the presence of 8-N3-Kdo and 7-N3-Kdo (respectively) compared to untreated cells, with differences also seen for single-cell gliding motility and type IV pilus-dependent swarm community expansion. While the surfaces of 8-N3-Kdo-grown cells were fluorescently labeled following treatment with dibenzocyclooctyne-linked fluorophores, the surfaces of 7-N3-Kdo-grown cells could not undergo fluorescent tagging. Activity analysis of the KdsB enzyme required to activate Kdo prior to its integration into nascent LPS molecules revealed that while 8-N3-Kdo is indeed a substrate of the enzyme, 7-N3-Kdo is not. Though a lack of M. xanthus cell aggregation was shown to expedite growth in liquid culture, 7-N3-Kdo-grown cells did not manifest differences in intrinsic clumping relative to untreated cells, suggesting that 7-N3-Kdo may instead be catabolized by the cells. Ultimately, these data provide important insights into the synthesis and cellular processing of valuable metabolic labels and establish a basis for the elucidation of fundamental principles of OM dynamism in live bacterial cells.

Dataset Information

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

Publications

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

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