title: Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86).
abstract: The moderate thermophile Bacillus stearothermophilus was used as a host in which to detect more thermostable variants of the B.pumilus chloramphenicol acetyltransferase (Cat-86) protein. Seventeen mutants were isolated and detected by their ability to grow in the presence of chloramphenicol at a previously restrictive temperature (58 degrees C). The genes encoding these proteins were sequenced; all 17 mutants carried the same C to T transition that conferred an amino acid substitution of alanine by valine at position 203 of the protein sequence. The wild-type and one mutant Cat-86 protein were purified to homogeneity using affinity chromatography, and kinetic and thermal stability studies were undertaken. Both enzymes had similar sp. act. in the region of 215 U/mg, with Km values for chloramphenicol in the range 13.8-15.4 microM and for acetyl CoA in the range 13.6-15.5 microM. The A203V mutant shows greater stability than the wild-type Cat-86 protein at temperatures above 50 degrees C and appears to pass through a transition state between 48 and 50 degrees C.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
authors.forename: S L
authors.initials: SL
authors.lastname: Turner
authors.forename: G C
authors.initials: GC
authors.lastname: Ford
authors.forename: A
authors.initials: A
authors.lastname: Mountain
authors.forename: A
authors.initials: A
authors.lastname: Moir
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D003198:Computer Simulation; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D015500:Chloramphenicol O-Acetyltransferase