Identifying Motifs for Phosphatidylcholine Activation of Bacterial Phosphatidylinositol-Specific Phospholipase C Enzymes

Abstract

Phosphatidylinositol-specific phospholipase Cs (PI-PLCs) secreted by pathogenic bacteria are often virulence factors. For one well-studied example, Bacillus thuringiensis PI-PLC (BtPI-PLC), the presence of the non-substrate lipid phosphatidylcholine (PC) in membranes enhances both membrane binding and enzymatic activity towards phosphatidylinositol (PI). A strip of four surface-exposed Tyr residues near the rim of the α/β-barrel are involved in this enhancement, and it has been proposed that this strip contributes to a specific PC binding site. The related PI-PLC from Staphylococcus aureus (SaPI-PLC), has similar kinetic characteristics to BtPI-PLC, but has only two Tyr residues in the same region. While SaPI-PLC can also be specifically activated towards PI cleavage by incorporation of PC into assay systems, SaPI-PLC membrane binding as a function of PC content is considerably weaker than that of BtPI-PLC. Mutagenesis of the two SaPI-PLC Tyr residues (Y253S/Y255S) reduces interfacial activity of SaPI-PLC. Adding the two ‘missing’ Tyr residues (N254Y/H258Y) did not further enhance SaPI-PLC specific activity but it dramatically enhanced binding of the protein to PC-rich vesicles. This suggests that PC activation is more complicated than simply binding the protein to membranes. Comparing enzyme kinetics and the effects of mutations on BtPI-PLC, SaPI-PLC and the PI-PLC from an intracellular pathogen (Listeria monocytogenes) suggests a rationale for the acquisition of a discrete PC binding site in some, but not all, PI-PLC enzymes.