Biochemical and Functional Characterization of an Iron-Containing Alcohol Dehydrogenase from Thermococcus barophilus Ch5.

Two iron-containing alcohol dehydrogenases (ADHs) are encoded in the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba ADH and Tba ADH). In our previous publication, we reported biochemical characteristics and catalytic mechanism of Tba ADH. Herein, we present evidence that Tba ADH possesses two activities for ethanol oxidization and acetaldehyde reduction at high temperature, capable of using NAD(H) and NADP(H) as coenzyme. Biochemical data show that Tba ADH possesses optimal reaction temperature, thermostability, divalent ion requirement, and substrate specificity distinct from Tba ADH and other iron-containing ADH homologues. However, Tba ADH and Tba ADH display same optimal reaction pH. Kinetic analyses demonstrate that Tba ADH displays higher catalytic efficiency for acetaldehyde reduction than that for ethanol oxidation, which is consistent with Tba ADH. Mutational data demonstrate that residues D115, K118, E159, D190, and E215 in Tba ADH, which has not been described to date, are necessary for enzyme activity, thus augmenting our understanding on catalytic mechanism of iron-containing ADH. Overall, our work demonstrates that Tba ADH is an iron-containing ADH with novel features, which is distinct from Tba ADH, thus providing a potential biocatalyst for biotransformation reaction.