A continuous fluorescent enzyme assay for early steps of lipid A biosynthesis
- 作者：Ronald J. Jenkins ; Garry D. Dotson ; gdotson@umich.edu
- 关键词：Lipid A ; Acyltransferase ; Fluorescence assay ; Acyl carrier protein ; ThioGlo
- 刊名：Analytical Biochemistry
- 出版年：2012
- 期刊代码：93_00032697
- 类别：imm
- 出版时间：1 June, 2012
- 卷：425
- 期：1
- 页码：21-27
- 文件大小：594 K
摘要
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 位ex = 379 nm and 位em = 513 nm using a microtiter plate reader. This assay marks the first continuous and nonradioactive assay for either acyltransferase.