NDLI: Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP

Content Provider	PubMed Central
Author	Demendi, Melinda Ishiyama, Noboru Lam, Joseph S. Berghuis, Albert M. Carole, Creuzenet
Copyright Year	2005
Abstract	WbpP is the only genuine UDP-GlcNAc (UDP-N-acetylglucosamine) C4 epimerase for which both biochemical and structural data are available. This represents a golden opportunity to elucidate the molecular basis for its specificity for N-acetylated substrates. Based on the comparison of the substrate binding site of WbpP with that of other C4 epimerases that convert preferentially non-acetylated substrates, or that are able to convert both acetylated and non-acetylated substrates equally well, specific residues of WbpP were mutated, and the substrate specificity of the mutants was determined by direct biochemical assays and kinetic analyses. Most of the mutations tested were anticipated to trigger a significant switch in substrate specificity, mostly towards a preference for non-acetylated substrates. However, only one of the mutations (A209H) had the expected effect, and most others resulted in enhanced specificity of WbpP for N-acetylated substrates (Q201E, G102K, Q201E/G102K, A209N and S143A). One mutation (S144K) totally abolished enzyme activity. These data indicate that, although all residues targeted in the present study turned out to be important for catalysis, determinants of substrate specificity are not confined to the substrate-binding pocket and that longer range interactions are essential in allowing proper positioning of various ligands in the binding pocket. Hence prediction or engineering of substrate specificity solely based on sequence analysis, or even on modelling of the binding pocket, might lead to incorrect functional assignments.
Related Links	http://dx.doi.org/10.1042/BJ20050263
Starting Page	173
File Format	PDF
ISSN	14708728
e-ISSN	14708728
Journal	Biochemical Journal
Issue Number	Pt 1
Volume Number	389
Language	English
Publisher	Portland Press Ltd.
Publisher Date	2005-07-01
Access Restriction	Open
Rights Holder	Portland Press Ltd.
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology Biochemistry

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