Over-production, purification and properties of the uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli.
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.
Liger
D
D
Unité de Recherche Associée 1131 du Centre National de la Recherche Scientifique, Université Paris-Sud, France.
Masson
A
A
Blanot
D
D
van Heijenoort
J
J
Parquet
C
C
D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000096:biosynthesis / Q000302:isolation & purification*; D013379:Substrate Specificity; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000378:metabolism*
D014538:Uridine Diphosphate N-Acetylmuramic Acid; D010453:Peptide Synthases; C023229:UDP-N-acetylmuramoyl-alanine synthetase; D000409:Alanine