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.
Auger
Geneviève
G
Enveloppes Bactériennes et Antibiotiques, UMR 8619 du CNRS, Bâtiment 430, Université de Paris-Sud, 91405, Orsay, France.
van Heijenoort
Jean
J
Mengin-Lecreulx
Dominique
D
Blanot
Didier
D
D001425:Bacterial Outer Membrane Proteins; D002851:Chromatography, High Pressure Liquid; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D007700:Kinetics; D009005:Monosaccharides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism; D015203:Reproducibility of Results; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D009005:Monosaccharides; D009842:Oligopeptides; D010457:Peptidoglycan; C438940:lipid I; D014537:Uridine Diphosphate N-Acetylglucosamine; D017351:N-Acetylglucosaminyltransferases; C064057:UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase; D004121:Dimethyl Sulfoxide