Topological and functional characterization of WbpM, an inner membrane UDP-GlcNAc C-6 dehydratase essential for lipopolysaccharide biosynthesis in Pseudomonas aeruginosa

Citation
C. Creuzenet et Js. Lam, Topological and functional characterization of WbpM, an inner membrane UDP-GlcNAc C-6 dehydratase essential for lipopolysaccharide biosynthesis in Pseudomonas aeruginosa, MOL MICROB, 41(6), 2001, pp. 1295-1310
Citations number
60
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
MOLECULAR MICROBIOLOGY
ISSN journal
0950-382X → ACNP
Volume
41
Issue
6
Year of publication
2001
Pages
1295 - 1310
Database
ISI
SICI code
0950-382X(200109)41:6<1295:TAFCOW>2.0.ZU;2-A
Abstract
WbpM is essential for the biosynthesis of B-band lipopolysaccharide (LPS) i n many serotypes of Pseudomonas aeruginosa. Homologues that can functionall y complement a wbpM null mutant and that are also necessary for virulence h ave been identified in numerous pathogenic bacteria. WbpM and most of its h omologues are large membrane proteins, which has long hampered the elucidat ion of their biochemical function. This paper describes the detailed charac terization of WbpM using both in vivo and in vitro approaches. LacZ and Pho A fusion experiments showed that WbpM was anchored to the inner membrane vi a four N-terminal transmembrane domains, whereas the C-terminal catalytic d omain resided in the cytoplasm. Although the membrane domains did not have any catalytic activity, complementation experiments suggested that they wer e 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 C-6 dehydratase specific for UDP-GlcNAc. It exhibi ted unusual low temperature (25-30 degreesC) and high pH (pH10) optima. Alt hough WbpM possessed an altered catalytic triad composed of SMK as opposed to SYK commonly found in other dehydratases, its catalysis was very efficie nt, with a k(cat) of 168 min(-1) and a k(cat)/K-m of 58 mM(-1) min(-1). The se unusual physico-kinetic properties suggested a potentially different mec hanism Of C6 dehydration for WbpM and its large homologues. His-S262 is now a precious tool for further structure-function studies.