Analysis of a conserved hydrophobic pocket important for the thermostability of Bacillus pumilus chloramphenicol acetyltransferase (CAT-86)
| Autor: | Haridasan Chirakkal, G.C. Ford, Anne Moir |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Chloramphenicol O-Acetyltransferase
Models Molecular Protein Folding Hot Temperature Mutant Bioengineering Bacillus Biochemistry Polymerase Chain Reaction Catalysis Conserved sequence Chloramphenicol acetyltransferase Protein structure Bacterial Proteins Consensus Sequence Enzyme Stability Computer Simulation Amino Acid Sequence Cloning Molecular Molecular Biology Conserved Sequence Thermostability chemistry.chemical_classification biology Bacillus pumilus biology.organism_classification Amino acid Protein Structure Tertiary Kinetics Enzyme chemistry Amino Acid Substitution Mutation Mutagenesis Site-Directed Biotechnology |
| Zdroj: | Protein engineering. 14(3) |
| ISSN: | 0269-2139 |
| Popis: | Site-directed mutagenesis was carried out on Bacillus pumilus chloramphenicol acetyltransferase (CAT-86) to determine the effects of substitution at a conserved hydrophobic pocket identified earlier as important for thermostability. Mutations were introduced that would substitute residues at consensus positions 33, 191 and 203 in the enzyme, both individually and in combination. Two mutants, SDM1 (CAT-86 Y33F, A203V) and SDM5 (CAT-86 A203I), were more thermostable than wild-type and two mutants, SDM4 (CAT-86 I191V) and SDM7 (CAT-86 A203G), were less stable. Reconstruction of the residues of this hydrophobic pocket to that of a more thermostable CAT-R387 enzyme pocket (as a Y33F, I191V, A203V triple mutant) increased the thermostability of the enzyme above the wild-type, but its stability was less than that of SDM1 and SDM5. The K(m) values of the mutant enzymes for chloramphenicol and acetyl-CoA were essentially unaltered (in the ranges 15-30 and 26-35 microM respectively) and the specific activity of purified enzyme was in the range 270-710 units/mg protein. The possible effects of the amino acid substitutions on the CAT-86 structure were determined by homology modelling. A reduction in conformational strain and optimized hydrophobic interactions are predicted to be responsible for the increased thermostability of the SDM1 and SDM5 mutants. |
| Databáze: | OpenAIRE |
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