BIOPHYSICAL AND MUTAGENIC ANALYSIS OF THERMOANAEROBACTER-ETHANOLICUS SECONDARY ALCOHOL-DEHYDROGENASE ACTIVITY AND SPECIFICITY

The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondar y-alcohol dehydrogenase (2 degrees ADH) was overexpressed in Escherich ia coli at more than 10%, of total protein. The recombinant enzyme was purified in high yield (67 %) by heat-treatment at 85 degrees C and ( NH4)(2)SO4 precipitation. Site-directed mutants (C37S, H59N, D150N, D1 50E and D150C were analysed to test the peptide sequence comparison-ba sed predictions of amino acids responsible for putative catalytic Zn b inding. X-ray absorption spectroscopy confirmed the presence of a prot ein-bound Zn atom with ZnS1(imid)(1)(N,O)(3) co-ordination sphere. Ind uctively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type 2 degrees ADH subunit. The C37S, H59N and D150N mu tant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectivel y, suggesting that these residues are involved in Zn liganding. The D1 50E and D150C mutants retained 0.47 and 1.2 Zn atoms per subunit, indi cating that an anionic side-chain moiety at this position preserves th e bound Zn. All five mutant enzymes had less than or equal to 3% of wi ld-type catalytic activity, suggesting that the T. ethanolicus 2 degre es ADH requires a properly co-ordinated catalytic Zn atom. The His-59 and Asp-150 mutations also altered 2 degrees ADH affinity for propan-2 -ol over a 140-fold range, whereas the overall change in affinity for ethanol spanned a range of only 7-fold, supporting the importance of t he metal in 2 degrees ADH substrate binding. The lack of significant c hanges in cofactor affinity as a result of these catalytic Zn ligand m utations suggested that 2 degrees ADH substrate- and cofactor-binding sites are structurally distinct. Altering Gly(198) to Asp reduced the enzyme specific activity 2.7-fold, increased the K-m(app) for NADP(+) 225-fold, and decreased the K-m(app) for NAD(+) 3-fold, supporting the prediction that the enzyme binds nicotinamide cofactor in a Rossmann fold. Our data indicate therefore that, unlike the liver 1 degrees ADH , the Rossmann-fold-containing T. ethanolicus 2 degrees ADH binds its catalytic Zn atom using a sorbitol dehydrogenase-like Cys-His-Asp moti f and does not bind a structural Zn atom.