Partial purification and specificity studies of the d‐glutamate‐adding and d‐alanyl‐d‐alanine‐adding enzymes from Escherichia coli K12

Abstract

The d-glutamate-adding and d-alanyl-d-alanine-adding enzymes from Escherichia coli were partially purified by fast protein liquid chromatography on an anion exchanger. Their relative molecular masses, determined by gel filtration on Superose 12, were 54000 ± 2000 and 51000 ± 2000, respectively. In order to investigate the specificity of these ligases, several compounds derived from their respective nucleotide substrates were prepared. In the case of the d-Glu-adding enzyme, DDP-MurNAc-l-Ala (DDP = dihydrouridine 5′-diphosphate) and P¹-MurNAc-l-Ala were substrates of the reaction. In the case of the d-Ala-d-Ala-adding enzyme, only DDP-MurNAc-l-Ala-d-Glu(-meso-A₂pm) was a subsrate; P¹-MurNAc-l-Ala-d-Glu(-meso-A₂pm) was neither a substrate nor an inhibitor. Concerning the amino acid site of the d-Glu-adding enzyme, even closely related analogues of d-glutamate hardly inhibited the reaction.