Altering substrate specificity of phosphatidylcholine-preferring phospholipase C of Bacillus cereus by random mutagenesis of the headgroup binding site.
PLC(Bc) is a 28.5 kDa monomeric enzyme that catalyzes the hydrolysis of the phosphodiester bond of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to provide a diacylglycerol and the corresponding phosphorylated headgroup. Because single replacements of Glu4, Tyr56, and Phe66 in the headgroup binding pocket led to changes in substrate specificity [Martin et al. (2000) Biochemistry 39, 3410-3415], a combinatorial library of approximately 6000 maltose binding protein-PLC(Bc) fusion protein mutants containing random permutations of these three residues was generated to identify PLC(Bc) mutants with altered specificity profiles and high catalytic activities. Members of this library were screened for hydrolytic activity toward the water soluble substrates C6PC, C6PE, and C6PS using a novel protocol that was conducted in a 96-well format and featured the in situ cleavage of the fusion protein to release the mutant PLC(Bc)s. Ten mutant enzymes that exhibited significant preferences toward C6PE or C6PS were selected and analyzed by steady-state kinetics to determine their specificity constants, k(cat)/K(M). The C6PS selective clones E4G, E4Q/Y56T/F66Y, and E4K/Y56V exhibited higher specificity constants toward C6PS than wt, whereas Y56T, F66Y, and Y56T/F66Y were C6PE selective and had comparable or higher specificity constants than wt for C6PE. The corresponding wt residues were singly reinserted back into the E4Q/Y56T/F66Y and E4K/Y56V mutants via site-directed mutagenesis, and the E4Q/F66Y mutant thus obtained exhibited a 10-fold higher specificity constant toward C6PS than wt, a value significantly higher than other PLC(Bc) mutants. On the basis of available data, 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. Thus, the substrate specificity of PLC(Bc) can be modulated by varying three of the amino acid residues that constitute the headgroup binding pocket, and it is now apparent that this enzyme is not evolutionarily optimized to hydrolyze phospholipids with ethanolamine or serine headgroups.
Antikainen
Nina M
NM
Department of Chemistry and Biochemistry and The Institute of Cellular and Molecular Biology, The University of Texas, Austin, Texas 78712, USA.
Hergenrother
Paul J
PJ
Harris
Micheleen M
MM
Corbett
William
W
Martin
Stephen F
SF
D019943:Amino Acid Substitution / Q000235:genetics; D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008823:Micelles; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D010713:Phosphatidylcholines / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
C107840:1,2-dihexanoic-sn-glycero-3-phosphoserine; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D008823:Micelles; D019151:Peptide Library; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D011993:Recombinant Fusion Proteins; C048072:1,2-dihexanoylphosphatidylethanolamine; D008320:Maltose; D010738:Type C Phospholipases