title: Study of the overproduced uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli.
abstract: The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli is responsible for the addition of the first amino acid of the peptide moiety in the assembly of the monomer unit of peptidoglycan. It catalyzes the formation of the amide bond between UDP-N-acetylmuramic acid (UDP-MurNAc) and L-alanine. The UDP-MurNAc-L-alanine ligase was overproduced 2000-fold in a strain harboring a recombinant plasmid (pAM1005) with the murC gene under the control of the inducible promoter trc. The murC gene product appears as a 50-kDa protein accounting for ca. 50% of total cell proteins. A two-step purification led to 1 g of a homogeneous protein from an 8-liter culture. The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the gene. The stability of the enzymatic activity is strictly dependent on the presence of 2-mercaptoethanol. The K(m) values for substrates UDP-N-acetylmuramic acid, L-alanine, and ATP were estimated; 100, 20, and 450 microM, respectively. The specificity of the enzyme for its substrates was investigated with various analogues. Preliminary experiments attempting to elucidate the enzymatic mechanism were consistent with the formation of an acylphosphate intermediate.
authors.affiliation: Unité de Recherche Associée 1131 du CNRS, Biochimie Moléculaire et Cellulaire, Université Paris-Sud, France.
authors.forename: D
authors.initials: D
authors.lastname: Liger
authors.forename: A
authors.initials: A
authors.lastname: Masson
authors.forename: D
authors.initials: D
authors.lastname: Blanot
authors.forename: J
authors.initials: J
authors.lastname: van Heijenoort
authors.forename: C
authors.initials: C
authors.lastname: Parquet
mesh_terms: D000409:Alanine / Q000378:metabolism; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D007700:Kinetics; D010453:Peptide Synthases / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D013379:Substrate Specificity
chemical_list: D010453:Peptide Synthases; C023229:UDP-N-acetylmuramoyl-alanine synthetase; D000409:Alanine