Analysis of a conserved hydrophobic pocket important for the thermostability of Bacillus pumilus chloramphenicol acetyltransferase (CAT-86)

Site-directed mutagenesis was carried out on Bacillus pumilus chloramphenic ol acetyltransferase (CAT-86) to determine the effects of substitution at a conserved hydrophobic pocket identified earlier as important for thermosta bility, Mutations were introduced that would substitute residues at consens us positions 33, 191 and 203 in the enzyme, both individually and in combin ation, 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 th is hydrophobic pocket to that of a more thermostable CAT-R387 enzyme pocket (as a Y33F, I191V, A203V triple mutant) increased the thermostability of t he 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 ace tyl-CoA were essentially unaltered (in the ranges 15-30 and 26-35 muM respe ctively) 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 predict ed to be responsible for the increased thermostability of the SDM1 and SDM5 mutants.