Biophysical and mutagenic analysis of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase activity and specificity

The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (2 degrees ADH) was overexpressed in Escherichia 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, D150E and D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding. X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS1(imid)(1)(N,O)(3) co-ordination sphere. Inductively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type 2 degrees ADH subunit. The C37S, H59N and D150N mutant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectively, suggesting that these residues are involved in Zn liganding. The D150E and D150C mutants retained 0.47 and 1.2 Zn atoms per subunit, indicating that an anionic side-chain moiety at this position preserves the bound Zn. All five mutant enzymes had less than or equal to 3% of wild-type catalytic activity, suggesting that the T. ethanolicus 2 degrees 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 the metal in 2 degrees ADH substrate binding. The lack of significant changes in cofactor affinity as a result of these catalytic Zn ligand mutations 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 motif and does not bind a structural Zn atom.