W0025

Structural Comparison of Two Different UDP-Glcnac-Modifying Short-Chain Dehydrogenase/Reductase Enzymes. Noboru Ishiyama¹, Carole Creuzenet², Joseph S. Lam³, Albert M. Berghuis¹, ¹Dept. of Biochemistry, McGill Univ., Montreal, QC, Canada, ²Dept. of Microbiology and Immunology, Univ. of Western Ontario, London, ON, Canada, ³Dept. of Microbiology, University of Guelph, Guelph, ON, Canada.

Pseudomonas aeruginosais an opportunistic bacterial pathogen that represents a major threat tocystic fibrosis patients. In the serotype O6, the early stages of biosynthesis of the B-band lipopolysaccharide, the major virulence factor in P. aeruginosa, are controlled by two UDP-GlcNAc-modifying enzymes, WbpP and WbpM. These enzymes are members of the short-chain dehydrogenase/reductase (SDR) superfamily. WbpP is a soluble C4 epimerase and WbpM is a membrane-anchored bifunctional C6 dehydratase/C4 reductase.

We have determined the three-dimensional structures of WbpP and the Helicobacter pylori enzyme FlaA1, a structural and functional homolog of the catalytic domain of WbpM. Diffraction datasets have been collected for four crystal complexes at NSLS (Upton, NY). The structures of the WbpP•NAD⁺•UDP-GalNAc, WbpP•NAD⁺•UDP-Glc, FlaA1•NADP⁺•UDP-GlcNAc and FlaA1•NADP⁺•UDP-Glc complexes have been determined at 2.0, 2.4, 2.7 and 2.8 Å resolution, respectively. While the structures of WbpP and FlaA1 reveal that both enzymes possess highly conserved SDR features, FlaA1 has a unique C-terminal fold among the currently known structures of the SDR enzymes.  Moreover, the comparison between the structures of WbpP and UDP-Glc 4-epimerases has suggested that the slightly modified sugar-binding pocket of WbpP may be responsible for its genuine UDP-GlcNAc 4-epimerase activity.  Likewise, dissimilarities found in the active sites of FlaA1 and other dehydratases in the SDR superfamily may provide key information that explains the bifunctional mechanism of FlaA1. These studies will hopefully reveal the molecular basis for the substrate specificity and catalytic mechanisms of WbpP and WbpM.