title: A continuous fluorescent enzyme assay for early steps of lipid A biosynthesis.
abstract: UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(R-3-hydroxyacyl)-glucosamine acyltransferase (LpxD) catalyze the first and third steps of lipid A biosynthesis, respectively. Both enzymes have been found to be essential for survival among gram-negative bacteria that synthesize lipopolysaccharide and are viable targets for antimicrobial development. Catalytically, both acyltransferases catalyze an acyl-acyl carrier protein (ACP)-dependent transfer of a fatty acyl moiety to a UDP-glucosamine core ring. Here, we exploited the single free thiol unveiled on holo-ACP after transfer of the fatty acyl group to the glucosamine ring using the thiol-specific labeling reagent, ThioGlo. The assay was continuously monitored as a change in fluorescence at λ(ex)=379 nm and λ(em)=513 nm using a microtiter plate reader. This assay marks the first continuous and nonradioactive assay for either acyltransferase.
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, 48109, USA.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Jenkins
authors.forename: Garry D
authors.initials: GD
authors.lastname: Dotson
mesh_terms: D000217:Acyltransferases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002384:Catalysis; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D005453:Fluorescence; D008050:Lipid A / Q000096:biosynthesis*
chemical_list: D001426:Bacterial Proteins; D008050:Lipid A; D000217:Acyltransferases; C050304:acyl-(acyl-carrier-protein)-UDP-N-acetylglucosamine acyltransferase