Altering and examining the substrate specificity of phospholipase C from bacillus cereus

textGenerating mutant enzymes that are selective for one substrate over others represents an important goal of modern protein engineering. Structural and thermodynamic studies of such mutant enzymes and their complexes with substrate analogues can lead to a better understanding of the structural basis for substrate selectivity. In this context the Martin group has focused a series of efforts toward developing methodology for altering the substrate selectivity of the phosphatidylcholine preferring phospholipase C from Bacillus cereus (PLCBc). PLCBc is a 28.5 kDa monomeric enzyme that catalyzes the hydrolysis of the phosphodiester bond of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. To identify PLCBc mutants with altered specificity profiles and catalytic activities similar to wild-type PLCBc, a combinatorial library of approximately 4000 maltose binding protein-PLCBc fusion protein mutants containing random permutations of the residues in the substrate binding site (Glu4, Tyr56, and Phe66) was generated. Members of this library were screened for hydrolytic activity toward the water soluble substrates 1,2-dihexanoyl-snglycero-3-phosphocholine (C6PC), 1,2-dihexanoyl-sn-glycero-3- phosphoethanolamine (C6PE), and 1,2-dihexanoyl-sn-glycero-3-phospho-L-serine (C6PS) using a colorimetric screen conducted in 96-well format. Ten mutant enzymes that exhibited significant preferences toward C6PE or C6PS were selected and analyzed by steady-state kinetics. The corresponding wt residues were singly reinserted back into two selected enzymes, E4Q/Y56T/F66Y and E4K/Y56V, via site-directed mutagenesis, to assess the impact of the specific substitutions in the mutants. The utility of the combinatorial approach described is supported by the observation that the most selective PLCBc mutants identified were either double or triple mutants. An aromatic residue at position 66 appears important for significant catalytic activity toward all three substrates, especially C6PC and C6PE. The charge of residue 4 also appears to be a determinant of enzyme specificity as a negatively charged residue at this position endows the enzyme with C6PC and C6PE preference, whereas a polar neutral or positively charged residue results in C6PS selectivity. Replacing Tyr56 with Val, Ala, Thr or Ser greatly reduces activity toward C6PC. The three-dimensional structures of the PLCBc mutants E4Q/F66Y, Y56V, and F66W were determined by X-ray crystallography, and only minor differences were observed between these structures and wild type PLCBc.Chemistry and BiochemistryChemistr