Biosynthesis of Peptidoglycan

The microenvironment of undecaprenyl diphosphateMurNAc-Ala-DGlu-Lys[N’-dimethylaminonaphthalene sulfonyl (Dns)]-rda-DAla in membrane fragments from Staphylococcus aureus Copenhagen was characterized by the fluorescent properties of the dansyl moiety of this lipid intermediate. These properties are the result of the enzymatic transfer of phospho-MurNAc-(NE-Dnsjpentapeptide from UDP-MurNAc-W-Dnsjpentapeptide to membrane-associated undecaprenyl phosphate. The emission maximum, quantum yield, and fluorescence lifetime of the dansylated lipid intermediate indicate that the fluorophore experiences a hydrophobic environment. The availability of the fluorophore to the lipid matrix is demonstrated by the paramagnetic quenching of fluorescence by the N-oxyl-4’,4’-dimethyloxazolidine derivative of 5-ketostearic acid that is intercalated into the membrane. The quenching efficiencies of a series of nitroxyl stearate derivatives, which differ with respect to the depth of the nitroxide within the membrane, suggest that the dansyl moiety of the lipid intermediate is close to the surface of the membrane. This conclusion is supported by the quenching efficiency observed with N,Ndiethyl-N-hexadecyl-N-tempoyl ammonium bromide, a nitroxide that is located at the membrane interface. This proximity to the membrane surface is not reflected in the availability of the fluorophore to N-methyl picolinium perchlorate in the aqueous phase. Only 23% of the dansyl fluorophores are available to quenching by this compound. A large value of anisotropy (0.32) and a high rotational relaxation time (>lOO ns) for the membrane-associated undecaprenyl diphosphate-MurNAc-(N’-Dnsjpentapeptide suggest that the lipid intermediate is immobilized within the membrane. The anisotropy of the dansylated lipid intermediate is not affected by changing the fluidity of the lipid matrix. These data suggest that undecaprenyl diphosphate MurNAcpentapeptide is immobilized within a hydrophobic environ-