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

Abstract

The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (secondary 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)2SO4 precipitation. Site-directed mutants (C37S, H59N, D150N, D150Eand 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 secondary 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 </= 3% of wild-type catalytic activity, suggesting that the T. ethanolicus secondary ADH requires a properly co-ordinated catalytic Zn atom. The His-59 and Asp-150 mutations also altered secondary 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 secondary ADH substrate binding. The lack of significant changes in cofactor affinity as a result of these catalytic Zn ligand mutations suggested that secondary ADH substrate-and cofactor-binding sites are structurally distinct. Altering Gly198 to Asp reduced the enzyme specific activity 2.7-fold, increased the Km(app) for NADP+ 225-fold, and decreased the Km(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 primary ADH,the Rossmann-fold-containing T. ethanolicus secondary ADH binds its catalytic Zn atom using a sorbitol dehydrogenase-like Cys-His-Asp motif and does not bind a structural Zn atom.

A Single Point Mutation Reverses the Enantiopreference of Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase

“…[16] Site-directed mutagenesis of T. ethanolicus adhB was performed in a two-step PCR approach as described [17] using the Expand High Fidelity PCR System (Roche), with plasmid pADHB1M1-kan as the template. [16] Site-directed mutagenesis of T. ethanolicus adhB was performed in a two-step PCR approach as described [17] using the Expand High Fidelity PCR System (Roche), with plasmid pADHB1M1-kan as the template.…”

Cloning, sequencing and expression inEscherichia coliof the primary alcohol dehydrogenase gene fromThermoanaerobacter ethanolicusJW200

“…The 2° Adh cloned from T. ethanolicus has been reported as differing in only three residues from T. brockii 2° Adh and as being 75% identical to a 2° Adh from the Clostridium beijerinckii [6,7]. However, T. ethanolicus AdhA is more similar to alcohol dehydrogenases from other species than to T. ethanolicus 2° Adh.…”

Extremophiles and their Application to Biofuel Research

“…A primary alcohol dehydrogenase (oxidation of primary alcohols; adhA) appears to function primarily during ethanol consumption, while a secondary alcohol dehydrogenase (oxidation of secondary alcohols; adhB) functions in ethanol production. A adhA strain was reported to display high alcohol tolerance (8% v/v) compared to the wild type (<2% v/v), apparently due to a loss of ethanol consumption and an acquired ability to accumulate C 30 fatty acids in the membrane (Burdette et al, 1997(Burdette et al, ,2002.…”

“…Thermoanaerobacter and Geobacillus are known to possess several ADHs, the study of which has assisted discrimination of specific activities (i.e., ethanol production and consumption, secondary alcohol production, etc.) (Lamed and Zeikus, 1981;Arni et al, 1996;Burdette et al, 1997Burdette et al, ,2002Talarico et al, 2005;Jeon et al, 2008;Peng et al, 2008). Examples from archaea are less common, but notable enzymes include those reported for Sulfolobussolfataricus (Ma and Adams, 2001;Ohshima et al, 2001;Raia et al, 2001;Esposito et al, 2002) and Thermococcushydrothermalis (Antoine et al, 1999), most being allkaliphilic and thermostable with high-temperature optima Secundo et al, 2005).…”