Characterization of a novel type III alcohol dehydrogenase from Thermococcus barophilus Ch5.

The genome of the hyperthermophilic and piezophilic euryarchaeaon Thermococcus barophilus Ch5 encodes three putative alcohol dehydrogenases (Tba ADHs). Herein, we characterized Tba ADH biochemically and probed its catalytic mechanism by mutational studies. Our data demonstrate that Tba ADH can oxidize ethanol and reduce acetaldehyde at high temperature with the same optimal temperature (75 °C) and exhibit similar thermostability for oxidization and reduction reactions. However, Tba ADH has different optimal pH for oxidation and reduction: 8.5 for oxidation and 7.0 for reduction. Tba ADH is dependent on a divalent ion for its oxidation activity, among which Mn is optimal. However, Tba ADH displays about 20% reduction activity without a divalent ion, and the maximal activity with Fe. Furthermore, Tba ADH showcases a strong substrate preference for 1-butanol and 1-hexanol over ethanol and other alcohols. Similarly, Tba ADH prefers butylaldehyde to acetaldehyde as its reduction substrate. Mutational studies showed that the mutations of residues D195, H199, H262 and H274 to Ala result in the significant activity loss of Tba ADH, suggesting that residues D195, H199, H262 and H274 are responsible for catalysis. Overall, Tba ADH is a thermoactive ADH with novel biochemical characteristics, thereby allowing this enzyme to be a potential biocatalyst.