The genome of the hyperthermophilic and piezophilic euryarchaeaon Thermococcus barophilus Ch5 encodes three putative alcohol dehydrogenases (Tba ADHs). Herein, we characterized Tba ADH547 biochemically and probed its catalytic mechanism by mutational studies. Our data demonstrate that Tba ADH547 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 ADH547 has different optimal pH for oxidation and reduction: 8.5 for oxidation and 7.0 for reduction. Tba ADH547 is dependent on a divalent ion for its oxidation activity, among which Mn2+ is optimal. However, Tba ADH547 displays about 20% reduction activity without a divalent ion, and the maximal activity with Fe2+. Furthermore, Tba ADH547 showcases a strong substrate preference for 1-butanol and 1-hexanol over ethanol and other alcohols. Similarly, Tba ADH547 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 ADH547, suggesting that residues D195, H199, H262 and H274 are responsible for catalysis. Overall, Tba ADH547 is a thermoactive ADH with novel biochemical characteristics, thereby allowing this enzyme to be a potential biocatalyst.
Keywords: Alcohol dehydrogenase; Biochemical characteristics; Hyperthermophilic Archaea.
Copyright © 2021 Elsevier B.V. All rights reserved.