A continuous fluorescent enzyme assay for early steps of lipid A biosynthesis

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.