title: A MurG assay which utilises a synthetic analogue of lipid I.
abstract: 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.
authors.affiliation: Enveloppes Bactériennes et Antibiotiques, UMR 8619 du CNRS, Bâtiment 430, Université de Paris-Sud, 91405, Orsay, France.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Auger
authors.forename: Jean
authors.initials: J
authors.lastname: van Heijenoort
authors.forename: Dominique
authors.initials: D
authors.lastname: Mengin-Lecreulx
authors.forename: Didier
authors.initials: D
authors.lastname: Blanot
mesh_terms: 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
chemical_list: 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