Topological and functional characterization of WbpM, an inner membrane UDP-GlcNAc C6 dehydratase essential for lipopolysaccharide biosynthesis in Pseudomonas aeruginosa.
WbpM is essential for the biosynthesis of B-band lipopolysaccharide (LPS) in many serotypes of Pseudomonas aeruginosa. Homologues that can functionally complement a wbpM null mutant and that are also necessary for virulence have been identified in numerous pathogenic bacteria. WbpM and most of its homologues are large membrane proteins, which has long hampered the elucidation of their biochemical function. This paper describes the detailed characterization of WbpM using both in vivo and in vitro approaches. LacZ and PhoA fusion experiments showed that WbpM was anchored to the inner membrane via four N-terminal transmembrane domains, whereas the C-terminal catalytic domain resided in the cytoplasm. Although the membrane domains did not have any catalytic activity, complementation experiments suggested that they were important for the polymerization of high-molecular-weight B-band LPS. The biochemical characterization of a soluble truncated form of WbpM, His-S262, showed that WbpM was a C6 dehydratase specific for UDP-GlcNAc. It exhibited unusual low temperature (25-30 degrees C) and high pH (pH 10) optima. Although WbpM possessed an altered catalytic triad composed of SMK as opposed to SYK commonly found in other dehydratases, its catalysis was very efficient, with a kcat of 168 min(-1) and a kcat/Km of 58 mM(-1) min(-1). These unusual physico-kinetic properties suggested a potentially different mechanism of C6 dehydration for WbpM and its large homologues. His-S262 is now a precious tool for further structure-function studies.
Creuzenet
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Department of Microbiology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
Lam
J S
JS
D001426:Bacterial Proteins; D001483:Base Sequence; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005816:Genetic Complementation Test; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D008070:Lipopolysaccharides; D011993:Recombinant Fusion Proteins; D006836:Hydro-Lyases; C441972:wbpM protein, Pseudomonas aeruginosa