Modulation of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C activity by mutations in the...

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) activity is activated by binding to phosphatidylcholine (PC) surfaces.
• This activation is partly due to improved protein-interface binding, but dimerization at the membrane interface was also suggested.

Purpose of the Study:

• To investigate the role of specific tyrosine residues in the dimerization and interfacial activation of Bacillus thuringiensis PI-PLC.
• To assess the impact of mutating these tyrosine residues on enzyme function, phospholipid binding, and catalytic activity.

Main Methods:

• Site-directed mutagenesis was used to replace tyrosine residues with serine.
• Phospholipid binding assays, enzymatic activity assays (PI cleavage and cIP hydrolysis), and kinetic analyses were performed.
• Crystal structures of mutant proteins were determined.

Main Results:

• Mutations of two tyrosine residues had minor effects on enzyme activity.
• Removal of three or four tyrosine residues significantly weakened PC binding and reduced PI cleavage.
• These mutations also reduced PC activation of cIP hydrolysis and impaired enzyme activity in various assays.
• Structural analysis revealed that mutants with three or four tyrosine removals crystallized as monomers, unlike the dimer observed in the W47A/W242A mutant.

Conclusions:

• The reduced catalytic activity of PI-PLC mutants lacking multiple tyrosine residues is likely due to either an impaired specific PC binding site or reduced dimerization potential.
• These findings highlight the importance of specific tyrosine residues for both interfacial activation and dimerization of Bacillus thuringiensis PI-PLC.