A MurG assay which utilises a synthetic analogue of lipid I

A standard assay for the MurG enzyme using a lipid I analogue [MurNAc(N(epsilon)-dansylpentapeptide)-pyrophosphoryl (R,S)-alpha-dihydroheptaprenol] and radioactive UDP-N-acetylglucosamine was set up. A high concentration (35%) of dimethylsulfoxide was necessary for maximal activity. Separation and quantitation were accomplished by reverse-phase high performance liquid chromatography (HPLC) in isocratic conditions and on-line radioactivity detection, thereby providing a rapid and accurate assay. The kinetic parameters of the MurG reaction were determined; the reaction was shown to also catalyse the reverse reaction at a measurable rate. A lipid I analogue containing dihydroundecaprenol as the prenyl chain turned out to be a poor MurG substrate, presumably owing to aggregation.