Open AccessA MurG assay which utilises a synthetic analogue of lipid I
Author(s) -
Auger Geneviève,
Heijenoort Jean,
MenginLecreulx Dominique,
Blanot Didier
Publication year - 2003
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/s0378-1097(02)01203-x
Subject(s) - high performance liquid chromatography , substrate (aquarium) , chromatography , chemistry , biochemistry , biology , ecology
A standard assay for the MurG enzyme using a lipid I analogue [MurNAc( N ɛ ‐dansylpentapeptide)‐pyrophosphoryl ( R , S )‐α‐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.