Over‐production, purification and properties of the uridine diphosphate N‐acetylmuramoyl‐l‐alanine: d‐glutamate ligase from Escherichia coli

Abstract

The UDP-N-acetylmuramoyl-l-alanine: d-glutamate ligase of Escherichia coli was over-produced in strains that harbour recombinant plasmids bearing the mur D gene under the control of the lac or P_R 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 K_m values for UDP-N-acetylmuramoyl-l-alanine; d-glutamate and ATP/Mg²⁺ were estimated at 7.5, 55 and 138 μM, 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.