Optimization of the Synthesis of Acyl-PG for Further Study of the Enzyme PldB

Rebecca L. Rose, Vassar College ’11 and Prof. Teresa A. Garrett

The wealth of scientific knowledge regarding its genome and related biochemical pathways, coupled with the convenience of its growth patterns make Escherichia coli strains excellent for determining the relationships between genes, gene expression and cellular metabolites. Phospholipids constitute a major class of those cellular metabolites. The biochemistry and genetics for the biosynthesis of the major phospholipids, phosphatidylethanolamine, phosphatidylglycerol (PG), phosphatidic acid, and cardiolipin is well understood. We are interested in a minor lipid metabolite, acyl-phosphatidylglycerol (acyl-PG). The enzyme PldB, in a reaction in which the acyl chain from 2-acyl-lyso-phospatidylethanolamine is transferred to the head group of phosphatidylglycerol, catalyzes the formation of acyl-PG. Mass spectrometry analysis of a strain lacking PldB was found to contain measurable amounts of [M-H]- ions with an exact mass and collision induced decomposition products consistent with acyl-PG. Our goal was to establish an in vitro enzyme assay to monitor PldB activity in order to determine the pathway by which acyl-PG is made in the PldB deficient strain. The assay mixture containing membranes derived from a wild type E. coli MG 1655, 32P-labeled PG, Tris buffer, 2-acyl-lysoPE, and diethylether was incubated at 37° C for various times and monitored by thin layer chromatography (TLC). Initial results yielded a product that can be preliminarily identified as acyl-PG due to its co-migration on TLC with a standard. Subsequent assays were focused on modernizing the in vitro reaction by replacing the diethylether with detergent. Thirteen detergents were tested at concentrations ranging from 5-0.05%. The most promising detergents from this larger screen were CHAPS, 1-s-octyl-β-D-thioglucopyranoside, and octyl-β-glucoside. These three detergents yielded acyl-PG at concentrations ranging 0.1% to 0.5%. These detergents were subsequently compared at several time points. To optimize the detergent concentration assays with 1-s-octyl-β-D-thioglucopyranoside were performed. Optimal acyl-PG formation with time occurred with 0.001% detergent. While the specific activity of PldB is lower in the presence of 1-s-octyl-β-D-thioglucopyranoside- 1.7 nmol/min/mg vs. 5.9 nmol/min/mg with ether- current work focuses on optimizing the in vitro reaction through the addition of stabilizing agents such as glycerol, varying ionic strength and pH.