Amphomycin inhibits mannosylphosphoryldolichol synthesis by forming a complex with dolichylmonophosphate

Abstract

The inhibitory effect of the lipopeptide antibiotic amphomycin on the mechanism of mannosylphosphoryldolichol biosynthesis by calf brain rough endoplasmic reticulum membranes has been studied extensively. Calf brain rough endoplasmic reticulum membranes when incubated with varying concentrations of GDP-mannose in the presence and absence of amphomycin showed no significant difference in apparent Km for GDP-mannose (1.08 and 1.37 microM, respectively). However, the Vmax was reduced to 0.17 pmol/mg protein/min in the presence of amphomycin as compared with 1.86 pmol/mg protein/min in its absence. On the other hand, when mannosylphosphoryldolichol synthase activity was measured in the presence of amphomycin and as a function of dolichylmonophosphate (Dol-P) concentrations, the shape of the substrate velocity curve changed from a rectangular hyperbola to a sigmoid. The Hill coefficients (n) for this reaction were calculated to be 2.02 and 1.22 in the presence and absence of the antibiotic and the corresponding Km values for Dol-P were found to be 333 and 47.3 microM, respectively. In separate experiments when radiolabeled antibiotic was reacted with Dol-P in the presence of Ca2+, a complex was formed. The complex formation was dependent on both Ca2+ in the reaction mixture and fatty acid residue on the antibiotic. Similar complex formation was also observed with undecaprenylmonophosphate. No such complex, however, was formed with dolichylpyrophosphate, with undecaprenylpyrophosphate, or with their free alcohols (dolichol or undecaprenol). Furthermore, when an equimolar mixture of Dol-P and phosphatidylserine was reacted with the antibiotic under identical conditions, the complex formation was observed selectively with Dol-P. These data demonstrated that amphomycin interacted with the active site of the glycosyl-carrier lipid (Dol-P), thereby preventing its participation at the enzymatic reaction.