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

Two iron-containing alcohol dehydrogenases (ADHs) are encoded in the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba ADH₆₄₁ and Tba ADH₅₄₇). In our previous publication, we reported biochemical characteristics and catalytic mechanism of Tba ADH₅₄₇. Herein, we present evidence that Tba ADH₆₄₁ 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 ADH₆₄₁ possesses optimal reaction temperature, thermostability, divalent ion requirement, and substrate specificity distinct from Tba ADH₅₄₇ and other iron-containing ADH homologues. However, Tba ADH₆₄₁ and Tba ADH₅₄₇ display same optimal reaction pH. Kinetic analyses demonstrate that Tba ADH₆₄₁ displays higher catalytic efficiency for acetaldehyde reduction than that for ethanol oxidation, which is consistent with Tba ADH₅₄₇. Mutational data demonstrate that residues D115, K118, E159, D190, and E215 in Tba ADH₆₄₁, 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 ADH₆₄₁ is an iron-containing ADH with novel features, which is distinct from Tba ADH₅₄₇, thus providing a potential biocatalyst for biotransformation reaction.