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Characterization of kinetic parameters of the lysophospholipase PldB using a liquid chromatography mass spectrometry based in vitro assay

Headgroup‐acylated glycerophospholipids (HAGPLs), such as acyl phosphatidylglycerol (acyl PG) and N‐acyl phosphatidylethanolamine, are a class of low‐abundance lipids that may play a role in Escherichia coli cell division. Lysophospholipase L2, also known as PldB, is an acyltransferase found in E. coli known to catalyze the formation of acyl PG. We seek to fully understand the substrate specificity of PldB to ultimately determine what contributes to PldB being an acyltransferase versus a phospholipase. Previous research suggests that PldB catalyzes the transfer of the acyl chain from 2‐acyl lysophospholipids to phosphatidylglycerol to form acyl PG. In order to characterize PldB's substrate specificity, a six‐histidine tagged variant of PldB has been constructed and overexpressed using the pET overexpression system. Cells induced to overexpress PldB are lysed, membranes isolated and PldB solubilized from the membranes with high concentrations of potassium chloride. The solubilized PldB is then purified to homogeneity from the resulting mixture with Ni‐affinity chromatography followed by gel‐filtration chromatography. This purified PldB will be used to determine the Km and kcat for PldB's reaction with a variety of acyl donors and acceptors using a liquid chromatography mass spectrometry (LC/MS) based assay currently under development.