BIOCHEMICAL-EVIDENCE FOR THE FORMATION OF A COVALENT ACYL-PHOSPHATE LINKAGE BETWEEN UDP-N-ACETYLMURAMATE AND ATP IN THE ESCHERICHIA-COLI UDP-N-ACETYLMURAMATE-L-ALANINE LIPASE-CATALYZED REACTION

In the peptidoglycan biosynthesis pathway in Escherichia coli, UDP-N-a cetylmuramate:L-alanine ligase (MurC) catalyzes the formation of UDP-N -acetylmuramyl-L-alanine. A peptide bond is formed in this reaction an d an ATP molecule is hydrolyzed concomitantly to produce ADP and ortho phosphate. A biochemical approach was devised to elucidate the role of ATP in this reaction. A fusion construct pMAL::murC was prepared and the maltose binding protein-UDP-N-acetylmuramyl:L-alanine ligase fusio n protein was overproduced in E. coli/pMal::murC upon isopropyl beta-t hiogalactoside induction. The fusion protein was purified to greater t han or equal to 90% homogeneity by a single-step affinity chromatograp hy. Subsequently, the ligase was released from the maltose binding pro tein by proteolytic cleavage and was purified to greater than or equal to 95% homogeneity by an ion-exchange chromatographic step. The kinet ic parameters of the regenerated ligase are comparable to those of the purified native enzyme. This ligase was used to investigate the role that ATP plays in the formation of UDP-N-acetylmuramyl-L-alanine. UDP- N-acetyl[O-18] muramate (with O-18 located at the carboxylate function only) was prepared by a combination of chemical and enzymatic process es and was used as the substrate of the ligase to probe the reaction m echanism. All reaction products were purified and subjected to liquid chromatographic-mass spectrometric analysis. A single [O-18]oxygen was transferred from UDP-N-acetyl[O-18]muramate to the orthophosphate pro duced in the reaction. No [O-18]oxygen was detected in the adenosine n ucleotides recovered from the reaction. These results strongly suggest that this ligase-catalyzed peptide formation proceeds through an acti vated acyl-phosphate linkage during the reaction process. ATP therefor e assists in the process of the peptide bond formation by donating its gamma-phosphoryl group to activate the carboxyl group of UDP-N-acetyl muramic acid.