The UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase of Esckevichiu 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 K , values for UDP-N-acetylmuramoyl-L-alanine; D-glutamate and ATP/ Mg2+ were estimated at 7.5, 55 and 138 pM, respectively. Under the most optimal in v i m conditions determined, a turnover number of 931 min-' was estimated. When considering the plasmid-free parental strain, the copy number of the murD gene product was not more than 1000 . cell-'. . Owing to its high specificity and to its occurrence only in eubacteria, the D-glutamate-adding enzyme is a potential target for new antibacterial agents. It was initially partially purified from Staphylococcus aureus [3, 41. In Escherichiu coli, various studies of the D-glutamateadding enzyme were carried out with crude extracts [5 -81. However, recently it was partially purified (33-fold) and its specificity was studied with analogues of D-glutamate and its nucleotide substrate, UDP-MurNAc-L-Ala [9]. Furthermore, the murD gene coding for this ligase has been identified at 2min on the chromosome of E. coli, in a large cluster of genes over pbpB-envA that code for proteins involved in peptidogylcan biosynthesis or cell division [lo]. The nucleotide sequence of murD has been determined [Il, 121. It contains 1314 nucleotides which are translated into 438 amino acids, corresponding to a protein with a molecular mass of 46936 Da.In the present paper we report on the over-production, the purification to homogeneity and the study of several properties of the D-glutamate-adding enzyme from E. coli.
Twenty-four analogues of D-glutamic acid were tested as substrates or inhibitors of the D-glutamate-adding enzyme from Escherichia coli. The best substrates were, in decreasing order of specific activity, D-erythro-4-methylglutamic acid, D-erythro-3-methylglutamic acid, DL-homocysteic acid, (+/-)-trans-1-amino-3-carboxy-cyclopentanecarboxylic acid and (+/-)-trans-1-amino-3-carboxy-cyclohexanecarboxylic acid. Among the different stereoisomers, only the D-erythro isomers for methylglutamic acids, and the trans isomers for the cyclic analogs, were substrates. Apart from the D-erythro-3- and 4-methylglutamic acids and DL-homocysteic acid, none of the examined compounds significantly inhibited the addition of radioactive D-glutamic acid to UDP-N-acetylmuramyl-L-alanine.
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