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| - Biochemical Evidence for the Formation of a Covalent Acyl-Phosphate Linkagebetween UDP-N-Acetylmuramate and ATP in the Escherichia coliUDP-N-Acetylmuramate:l-Alanine Ligase-Catalyzed Reaction
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| - In the peptidoglycan biosynthesis pathway inEscherichia coli,UDP-N-acetylmuramate:l-alanine ligase (MurC) catalyzes the formation ofUDP-N-acetylmuramyl-L-alanine. A peptidebond isformed in this reaction and an ATP molecule is hydrolyzed concomitantlyto produce ADP andorthophosphate. A biochemical approach was devised to elucidatethe role of ATP in this reaction. Afusion construct pMAL::murC was prepared and the maltosebindingprotein−UDP-N-acetylmuramyl:l-alanine ligase fusion protein was overproduced in E.coli/pMal::murC upon isopropylβ-thiogalactosideinduction. The fusion protein was purified to ≥90% homogeneityby a single-step affinity chromatography.Subsequently, the ligase was released from the maltose bindingprotein by proteolytic cleavage and waspurified to ≥95% homogeneity by an ion-exchange chromatographic step.The kinetic parameters of theregenerated ligase are comparable to those of the purified nativeenzyme. This ligase was used to investigatethe role that ATP plays in the formation ofUDP-N-acetylmuramyl-l-alanine.UDP-N-acetyl[18O]muramate(with 18O located at the carboxylate function only) wasprepared by a combination of chemical andenzymatic processes and was used as the substrate of the ligase toprobe the reaction mechanism. Allreaction products were purified and subjected to liquidchromatographic−mass spectrometric analysis.A single [18O]oxygen was transferred fromUDP-N-acetyl[18O]muramate to theorthophosphate producedin the reaction. No [18O]oxygen was detected inthe adenosine nucleotides recovered from the reaction.These results strongly suggest that this ligase-catalyzed peptideformation proceeds through an activatedacyl-phosphate linkage during the reaction process. ATP thereforeassists in the process of the peptidebond formation by donating its γ-phosphoryl group to activate thecarboxyl group of UDP-N-acetylmuramicacid.
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