Determination of the MurD mechanism through crystallographic analysis of enzyme complexes

UDP -N-acetylmuramoyl- l-alanine: d-glutamate (MurD) ligase catalyses the addition of d-glutamate to the nucleotide precursor UDP -N-acetylmuramoyl- l-alanine (UMA). The crystal structures of three complexes of Escherichia coli MurD with a variety of substrates and products have been determined to high resolution. These include (1) the quaternary complex of MurD, the substrate UMA, the product ADP, and Mg 2+, (2) the quaternary complex of MurD, the substrate UMA, the product ADP, and Mn 2+, and (3) the binary complex of MurD with the product UDP -N-acetylmuramoyl- l-alanine- d-glutamate (UMAG). The reaction mechanism supported by these structures proceeds by the phosphorylation of the C-terminal carboxylate group of UMA by the γ-phosphate group of ATP to form an acyl-phosphate intermediate, followed by the nucleophilic attack by the amino group of d-glutamate to produce UMAG. A key feature in the reaction intermediate is the presence of two magnesium ions bridging negatively charged groups.