Abstract
Broad‐range, phosphatidylcholine‐preferring phospholipase C (PC‐PLCLM) from Listeria monocytogenes is one of the critical virulence factors mediating the dissolution of vacuolar membranes so that the intracellular pathogen can escape into the cytosol and spread to neighboring cells. We have expressed the enzyme in E. coli and characterized its kinetics with a variety of substrates. The amino acid sequence of PC‐PLCLM is highly homologous to Bacillus cereus PC‐PLCBC. Both are metalloenzymes with identical residues coordinating to the Zn2+ ligands. Intriguingly, the PC‐PLCLM exhibits an acidic pH optimum (pH5~6) compared to the well‐studied PC‐PLCBC which is more efficient at basic pH values. This is significant as PC‐PLCLM activity is needed to aid in Listeria escape from acidified vacuolar pH. The optimal efficiency at acidic pH results from dramatically reduced Km values but moderate changes in kcat at lower pH. In addition, unlike PLCBC, PC‐PLCLM can cleave sphingomyelin. Mechanistic details for the acidic pH optimum and substrate specificity are probed with mutagenesis and a combination of biochemical and biophysical approaches. This study is supported by the National Institutes of Health GM60418.
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