Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP.
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.
Demendi
Melinda
M
Department of Microbiology and Immunology, University of Western Ontario, London, ON, N6A 5C1, Canada.
Ishiyama
Noboru
N
Lam
Joseph S
JS
Berghuis
Albert M
AM
Creuzenet
Carole
C
D000107:Acetylation; D001665:Binding Sites; D002384:Catalysis; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014534:UDPglucose 4-Epimerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D014534:UDPglucose 4-Epimerase