title: Role of the amino acid invariants in the active site of MurG as evaluated by site-directed mutagenesis.
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mutants proteins, in which residues T15, H18, Y105, H124, E125, N127, N134, S191, N198, R260, E268, Q288 or N291 have been replaced by alanine, were obtained as the C-terminal His-tagged forms.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.
authors: Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619 du CNRS, Université Paris-Sud, Bâtiment 430, 91405 Orsay Cedex, France.
authors: Muriel
authors: M
authors: Crouvoisier
authors: Geneviève
authors: G
authors: Auger
authors: Didier
authors: D
authors: Blanot
authors: Dominique
authors: D
authors: Mengin-Lecreulx
mesh_terms: D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000596:Amino Acids / Q000737:chemistry*; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D002250:Carbon Radioisotopes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D014169:Transformation, Bacterial
chemical_list: D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D029968:Escherichia coli Proteins; D006639:Histidine; D017351:N-Acetylglucosaminyltransferases; C064057:UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase; D000409:Alanine