title: Characterization of the dehydratase WcbK and the reductase WcaG involved in GDP-6-deoxy-manno-heptose biosynthesis in Campylobacter jejuni.
abstract: The capsule of Campylobacter jejuni strain 81-176 comprises the unusual 6-deoxy-α-D-altro-heptose, whose biosynthesis and function are not known. In the present study, we characterized enzymes of the capsular cluster, WcbK and WcaG, to determine their role in 6-deoxy-altro-heptose synthesis. These enzymes are similar to the Yersinia pseudotuberculosis GDP-manno-heptose dehydratase/reductase DmhA/DmhB that we characterized previously. Capillary electrophoresis and MS analyses showed that WcbK is a GDP-manno-heptose dehydratase whose product can be reduced by WcaG, and that WcbK/WcaG can use the substrate GDP-mannose, although with lower efficiency than heptose. Comparison of kinetic parameters for WcbK and DmhA indicated that the relaxed substrate specificity of WcbK comes at the expense of catalytic performance on GDP-manno-heptose. Moreover, although WcbK/WcaG and DmhA/DmhB are involved in altro- versus manno-heptose synthesis respectively, the enzymes can be used interchangeably in mixed reactions. NMR spectroscopy analyses indicated conservation of the sugar manno configuration during catalysis by WcbK/WcaG. Therefore additional capsular enzymes may perform the C3 epimerization necessary to generate 6-deoxy-altro-heptose. Finally, a conserved residue (Thr(187) in WcbK) potentially involved in substrate specificity was identified by structural modelling of mannose and heptose dehydratases. Site-directed mutagenesis and kinetic analyses demonstrated its importance for enzymatic activity on heptose and mannose substrates.
authors.affiliation: Department of Microbiology and Immunology, Infectious Diseases Research Group, University of Western Ontario, London, ON, Canada, N6A 5C1.
authors.forename: Matthew
authors.initials: M
authors.lastname: McCallum
authors.forename: Gary S
authors.initials: GS
authors.lastname: Shaw
authors.forename: Carole
authors.initials: C
authors.lastname: Creuzenet
mesh_terms: D001483:Base Sequence; D016123:Campylobacter jejuni / Q000201:enzymology / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D006155:Guanosine Diphosphate Mannose / Q000031:analogs & derivatives* / Q000096:biosynthesis; D006836:Hydro-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D006155:Guanosine Diphosphate Mannose; C054776:guanosine diphosphate-4-deoxymannose; D010088:Oxidoreductases; D006836:Hydro-Lyases