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.
Skip Nav Destination
Article navigation
July 2005
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
June 21 2005
Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP
Melinda DEMENDI;
Melinda DEMENDI
*Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada, N6A 5C1
Search for other works by this author on:
Noboru ISHIYAMA;
Noboru ISHIYAMA
†Department of Biochemistry, McGill University, Montréal, QC, Canada, H3A 1A4
Search for other works by this author on:
Joseph S. LAM;
Joseph S. LAM
‡Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
Search for other works by this author on:
Albert M. BERGHUIS;
Albert M. BERGHUIS
†Department of Biochemistry, McGill University, Montréal, QC, Canada, H3A 1A4
§Department of Microbiology and Immunology, McGill University, Montréal, QC, Canada, H3A 1A4
Search for other works by this author on:
Carole CREUZENET
Carole CREUZENET
1
*Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada, N6A 5C1
1To whom correspondence should be addressed (email ccreuzen@uwo.ca).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
February 10 2005
Revision Received:
March 07 2005
Accepted:
March 08 2005
Accepted Manuscript online:
March 08 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 389 (1): 173–180.
Article history
Received:
February 10 2005
Revision Received:
March 07 2005
Accepted:
March 08 2005
Accepted Manuscript online:
March 08 2005
Citation
Melinda DEMENDI, Noboru ISHIYAMA, Joseph S. LAM, Albert M. BERGHUIS, Carole CREUZENET; Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP. Biochem J 1 July 2005; 389 (1): 173–180. doi: https://doi.org/10.1042/BJ20050263
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.