Liger, D, Masson, A, Blanot, D, van Heijenoort, J and Parquet, C (1995) Over-production, purification and properties of the uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli. Eur. J. Biochem. 230:80-7
The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli was over-produced in strains harbouring recombinant plasmids bearing the murC gene under the control of the lac or trc promoter. Plasmid pAM1005, in which the promoter and ribosome-binding site region of murC were removed and in which the gene was directly under the control of promoter trc, led to a 2000-fold amplification of the L-alanine-adding activity after induction by isopropyl-thio-beta-D-galactopyranoside. The murC gene product was visualized as a 50-kDa protein accounting for approximately 50% of the cell protein. A two-step purification led to 1 g of a homogeneous protein from an 18-1 culture. The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the murC gene. The presence of 2-mercaptoethanol and glycerol was essential for the stability of the enzyme. The Km values for UDP-N-acetylmuramic acid, L-alanine and ATP/Mg2+ were estimated at 100, 20 and 450 microM, respectively. Under the optimal in vitro conditions a turnover number of 928 min-1 was calculated and a copy number/cell of 600 could be roughly estimated. The specificity of the enzyme for its substrates was investigated with various analogues. The enzyme also catalysed the reverse reaction.
Alanine/metabolism; Amino Acid Sequence; Base Sequence; Enzyme Stability; Escherichia coli/enzymology; Molecular Sequence Data; Peptide Synthases/biosynthesis; Peptide Synthases/isolation & purification; Substrate Specificity; Uridine Diphosphate N-Acetylmuramic Acid/metabolism
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