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Functional modification of phosphatidylinositol‐specific phospholipase C from Bacillus cereus by computer modeling and site‐directed mutagenesis

Phosphatidylinositol‐specific phospholipase C (PI‐PLC) is a secreted enzyme that serves as a virulence factor in many Gram‐positive bacteria such as Listeria monocytogenes (Lm) and Bacillus cereus (Bc). While these enzymes have similar specific activities on phosphatidylinositol (PI), BcPI‐PLC exhibits a much stronger ability to cleave glycosylphosphatidylinositol‐anchored proteins (GAP) than LmPI‐PLC. An extra β‐stand (Vb) is present in many bacterial PI‐PLCs such as BcPI‐PLC and B. anthracis PI‐PLC, but is absent in LmPI‐PLC. According to the crystal structure, the Vb β‐strand supports the preceding loop residues in forming a groove in BcPI‐PLC needed for GAP binding. In our recent studies, we have shown that deletion of the Vb β‐strand results in loss of activity on GAP. To further analyze the functions of BcPI‐PLC and its role in bacterial pathogenesis, we are using computer modeling to design site‐specific mutants with the expectation that some mutants will have retained activity either on PI or on GAP only. Five residues involved in the groove and the Vb β‐strand (N175, W178, P179, D180, F184) were individually replaced by 2–4 other amino acids, resulting in a total of 13 mutants. Our preliminary results showed that N175Y had no significant activity change on PI and GAP, W178E lost both activities, and both D180Y and D180E lost PI activity but retained about 50% of WT activity on GAP. These results indicated that D180 is an interesting position for further residue replacement to obtain a mutant that exhibits activity only on GAP. Analysis of other mutants is currently underway.