Over-production, purification and properties of the uridine diphosphate N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli.
The UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase of Escherichia coli was over-produced in strains that harbour recombinant plasmids bearing the murD gene under the control of the lac or PR promoter. Purification to homogeneity was achieved by a two-step procedure from a 181-fold over-producing strain. The N-terminal sequence of the purified protein was determined and correlated with the nucleotide sequence of the murD gene. The purified activity was highly dependent on the concentration of potassium phosphate and Mg2+. The enzyme also catalysed the reverse reaction. The Km values for UDP-N-acetylmuramoyl-L-alanine; D-glutamate and ATP/Mg2+ were estimated at 7.5, 55 and 138 microM, respectively. Under the most optimal in vitro conditions determined, a turnover number of 931 min-1 was estimated. When considering the plasmid-free parental strain, the copy number of the murD gene product was not more than 1000.cell-1.
Pratviel-Sosa
F
F
Unité de Recherche Associée 1131 du Centre National de la Recherche Scientifique, Biochimie Moléculaire et Cellulaire, Université Paris-Sud, Orsay, France.
Mengin-Lecreulx
D
D
van Heijenoort
J
J
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D011994:Recombinant Proteins; D010453:Peptide Synthases; C053379:UDP-N-acetylmuramoylalanine-D-glutamate ligase