Determination of the MurD mechanism through crystallographic analysis of enzyme complexes

UDP-N-acetylmuramoyl-L-alanine:D-glutamate (MurD) ligase catalyses the addi tion of D-glutamate to the nucleotide precursor UDP-N-acetylmuramoyl-L-alan ine (UMA). The crystal structures of three complexes of Escherichia coli Mu rD with a variety of substrates and products have been determined to high r esolution. These include (1) the quaternary complex of MurD, the substrate UMA, the product ADP, and Mg2+, (2) the quaternary complex of MurD, the sub strate UMA, the product ADP, and Mn2+, and (3) the binary complex of MurD w ith the product UDP-N-acetylmwamoyl-L-alanine-D-glutamate (UMAG). The react ion mechanism supported by these structures proceeds by the phosphorylation of the C-terminal carboxylate group of UMA by the gamma-phosphate group of ATP to form an acyl-phosphate intermediate, followed by the nucleophilic a ttack by the amino group of D-glutamate to produce UMAG. A key feature in t he reaction intermediate is the presence of two magnesium ions bridging neg atively charged groups. (C) 1999 Academic Press.