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Role of the amino acid invariants in the active site of MurG as evaluated by site-directed mutagenesis
Authors:Crouvoisier Muriel  Auger Geneviève  Blanot Didier  Mengin-Lecreulx Dominique
Institution:Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619 du CNRS, Université Paris-Sud, Batiment 430, 91405 Orsay Cedex, France.
Abstract:To evaluate their role in the active site of the MurG enzyme from Escherichia coli, 13 residues conserved in the sequences of 73 MurG orthologues were submitted to site-directed mutagenesis. All these residues lay within, or close to, the active site of MurG as defined by its tridimensional structure Ha et al., Prot. Sci. 9 (2000) 1045-1052, and Hu et al., Proc. Natl. Acad. Sci. USA 100 (2003) 845-849]. Thirteen mutants proteins, in which residues T15, H18, Y105, H124, E125, N127, N134, S191, N198, R260, E268, Q288 or N291 have been replaced by alanine, were obtained as the C-terminal His-tagged forms. The effects of the mutations on the activity were checked: (i) by functional complementation of an E. coli murG mutant strain by the mutated genes; and (ii) by the determination of the steady-state kinetic parameters of the purified proteins. Most mutations resulted in an important loss of activity and, in the case of N134A, in the production of a highly unstable protein. The results correlated with the assigned or putative functions of the residues based on the tridimensional structure.
Keywords:Glycosyltransferase  Invariant amino acids  MurG  Peptidoglycan  Site-directed mutagenesis
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