CLONING AND EXPRESSION OF THE GENE ENCODING THE THERMOANAEROBACTER-ETHANOLICUS 39E SECONDARY-ALCOHOL DEHYDROGENASE AND BIOCHEMICAL-CHARACTERIZATION OF THE ENZYME

The adhB gene encoding Thermoanaerobacter ethanolicus 39E secondary-al cohol dehydrogenase (S-ADH) was cloned, sequenced and expressed in Esc herichia coli. The 1056 bp gene encodes a homotetrameric recombinant e nzyme consisting of 37.7 kDa subunits. The purified recombinant enzyme is optimally active above 90 degrees C with a half-life of approx. 1. 7 h at 90 degrees C. An NADP(H)-dependent enzyme, the recombinant S-AD H has 1400-fold greater catalytic efficiency in propan-2-ol oxidation than in ethanol oxidation. The enzyme was inactivated by chemical modi fication with dithionitrobenzoate (DTNB) and diethylpyrocarbonate, ind icating that Cys and His residues are involved in catalysis. Zinc was the only metal enhancing S-ADH reactivation after DTNB modification, i mplicating the involvement of bound zinc in catalysis. Arrhenius plots for the oxidation of propan-2-ol by the native and recombinant S-ADHs were linear from 25 to 90 degrees C when the enzymes were incubated a t 55 degrees C before assay. Discontinuities in the Arrhenius plots fo r propan-2-ol and ethanol oxidations were observed, however, when the enzymes were preincubated at 0 or 25 degrees C. The observed Arrhenius discontinuity therefore resulted from a temperature-dependent, cataly tically significant S-ADH structural change. Hydrophobic cluster analy sis comparisons of both mesophilic and thermophilic S-ADH and primary- versus S-ADH amino acid sequences were performed. These comparisons p redicted that specific proline residues might contribute to S-ADH ther mostability and thermophilicity, and that the catalytic Zn ligands are different in primary-alcohol dehydrogenases (two Cys and a His) and S -ADHs (Cys, His, and Asp).