Initial Membrane Reaction in Peptidoglycan Synthesis: LIPID DEPENDENCE OF PHOSPHO-N-ACETYLMURAMYL-PENTAPEPTIDE TRANSLOCASE (EXCHANGE REACTION)

Abstract

Abstract The initial membrane reaction in the biosynthesis of peptidoglycan is catalyzed by phospho-N-acetylmuramyl-pentapeptide translocase (UDP-MurNAc - l - Ala - d - γ-Glu - l - Lys - d - Ala - d - Ala:undecaprenyl - phosphate phospho - MurNAc-pentapeptide transferase). In addition to the transfer reaction, the enzyme catalyzes the exchange of [3H]uridine monophosphate with the uridine monophosphate moiety of UDP - MurNAc - pentapeptide. Lipid-dependent translocase was obtained in an inactive form by gel filtration of membranes from Staphylococcus aureus Copenhagen on agarose A-50m in the presence of 1% Triton X-100. The excluded fraction was depleted of undecaprenyl-diphosphate-MurNAc-pentapeptide (g95%) and partially depleted of undecaprenyl-phosphate (g75%). After removal of Triton X-100, the translocase was reactivated with a lipid extract from membranes of S. aureus Copenhagen. The translocase was partially reactivated (10 to 30%) by the addition of dioleoyl phosphatidylcholine, phosphatidylcholine (plant), phosphatidylglycerol, and, to a lesser extent, by phosphatidylinositol and phosphatidylethanolamine when added in the presence of 1 x 10-3 m sodium lauroyl sarcosinate. The optimal concentration of dioleoyl phosphatidylcholine was 1.2 x 10-4 m. Maximal reactivation of the exchange reaction required the addition of either 5 x 10-7 m undecaprenyl-diphosphate - MurNAc - pentapeptide or 1 x 10-6 m undecaprenyl-phosphate to lipid-dependent translocase in the presence of dioleoyl phosphatidylcholine and sodium lauroyl sarcosinate. Thus, for activity in the exchange reaction, the translocase requires a phosphatide and is markedly stimulated by the addition of either undecaprenyl-phosphate or undecaprenyl-diphosphate-MurNAc-pentapeptide. The implications of the results with the undecaprenyl derivatives are discussed in relation to proposed reaction mechanisms.