STUDY OF THE OVERPRODUCED URIDINE-DIPHOSPHATE-N-ACETYLMURAMATE-L-ALANINE LIGASE FROM ESCHERICHIA-COLI

The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli is respo nsible for the addition of the first amino acid of the peptide moiety in the assembly of the monomer unit of peptidoglycan. It catalyzes the formation of the amide bond between UDP-N-acetylmuramic acid (UDP-Mur NAc) and L-alanine. The UDPMurNAc-L-alanine ligase was overproduced 20 00-fold in a strain harboring a recombinant plasmid (pAM1005) with the murC gene under the control of the inducible promoter trc, The murC g ene product appears as a 50-kDa protein accounting for ca, 50% of tota l cell proteins. A two-step purification led to 1 g of a homogeneous p rotein from an 18-liter culture, The N-terminal sequence of the purifi ed protein correlated with the nucleotide sequence of the gene, The st ability of the enzymatic activity is strictly dependent on the presenc e of 2-mercaptoethanol. The K-m values for substrates UDP-N-acetylmura mic acid, L-alanine, and ATP were estimated: 100, 20, and 450 mu M res pectively. The specificity of the enzyme for its substrates was invest igated with various analogues, Preliminary experiments attempting to e lucidate the enzymatic mechanism were consistent with the formation of an acylphosphate intermediate.