Expression and site-directed mutagenesis of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus: probing the role of the active site Glu146.
A series of site-specific mutants of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) was prepared in which the glutamic acid residue at position 146 was replaced with glutamine, aspartic acid, histidine, and leucine to elucidate what role Glu146 might play in catalysis. An expression system for the native enzyme in Escherichia coli was first developed to provide PLCBc that was fused via an intervening factor Xa protease recognition sequence at its N-terminus to maltose binding protein (MBP). This MBP-PLCBc fusion protein was isolated at levels of 50-70 mg/L of culture; selective trypsin digestion of the MBP-PLCBc fusion protein followed by chromatographic purification yielded recombinant PLCBc at levels of ca. 10 mg/L. Polymerase chain reaction (PCR) mutagenesis on the PLCBc gene (plc) was then used to replace the Glu146 codon with those for glutamine (E146Q), aspartic acid (E146D), histidine (E146H), and leucine (E146L). The catalytic efficiency of the E146Q mutant was 1.6% that of native PLCBc, while the other mutants each possessed activities of 0.2-0.3% of the wild type. The kcat/Km vs pH profiles for both E146Q and native PLCBc have ascending acidic limbs, suggesting that Glu146 does not serve as the general base in the hydrolysis reaction. As measured by circular dichroism, all of the mutant proteins contained less helical structure and underwent denaturation at lower temperatures than the wild type in the order: wild type > E146Q > E146D approximately E146H approximately E146L. Atomic absorption analyses indicated that the mutant proteins also exhibited lower Zn2+ content than the wild type. Thus, the Glu146 residue in PLCBc stabilizes the secondary and tertiary structure of the enzyme and serves as a critical ligand for Zn2, but it does not appear to have any specific catalytic role.
Martin
S F
SF
Department of Chemistry and Biochemistry, University of Texas at Austin 78712, USA. sfmartin@mail.utexas.edu
Spaller
M R
MR
Hergenrother
P J
PJ
D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D063065:Organophosphonates / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D013379:Substrate Specificity; D013696:Temperature; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D063065:Organophosphonates; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D018698:Glutamic Acid; D010738:Type C Phospholipases