Purification and characterization of PldB, an Escherichia coli lysoglycerophospholipase

Abstract

Headgroup‐acylated glycerophospholipids (GPLs) are an under‐studied class of lipids found in the Escherichia coli cell membrane that are now known to impact cell division by determining potential cell division sites. One such headgroup‐acylated GPL, acyl phosphatidylglycerol (acyl PG), is produced by the enzymatic transfer of the acyl chain from 2‐acyl lysophosphoethanolamine (2‐acyl lysoPE) to the headgroup of phosphatidylglycerol (PG). The enzyme implicated in this trans esterification reaction is the E. coli membrane‐associated protein, PldB. Because PldB is involved in the production of acyl PG, knowledge of the biosynthetic reaction of PldB can provide insight to the mechanism by which other headgroup‐acylated GPLs are formed. To study its biosynthetic reaction, PldB was purified to prevent interference from other enzymes that may metabolize either its reactants or products. A six‐histidine tagged version of PldB was over‐expressed in E. coli. This membrane‐associated protein was removed from the membrane using high salt and then purified using nickel‐affinity chromatography followed by gel‐filtration chromatography. Current research is focused on tracking the activity of PldB at various stages in the purification. The activity being measured is the rate of deacylation of 14C 2‐acyl lysophosphatidylglycerol, which is metabolized the same way that 2‐acyl lysoPE is metabolized. Once the efficiency of the purification is obtained, the substrate specificity with regard to acyl chain length of the acyl donor and acyl acceptor of PldB will be assessed using the purified enzyme.