A novel alcohol dehydrogenase in the hyperthermophilic crenarchaeon Hyperthermus butylicus

Author:

Tse Ching1,Ma Kesen1ORCID

Affiliation:

1. Department of Biology University of Waterloo Waterloo Ontario Canada

Abstract

AbstractHyperthermus butylicus is a hyperthermophilic crenarchaeon that produces 1‐butanol as an end product. A thermostable alcohol dehydrogenase (ADH) must be present in H. butylicus to act as the key enzyme responsible for this production; however, the gene that encodes the ADH has not yet been identified. A novel ADH, HbADH2, was purified from a cell‐free extract of H. butylicus, and its characteristics were determined. The gene that encodes HbADH2 was demonstrated to be HBUT_RS04850 and annotated as a hypothetical protein in H. butylicus. HbADH2 was found to be a primary–secondary ADH capable of using a wide range of substrates, including butyraldehyde and butanol. Butyraldehyde had the highest specificity constant, calculated as kcat/Km, with kcat and apparent Km values of 8.00 ± 0.22 s−1 and 0.59 ± 0.07 mM, respectively. The apparent Km values for other substrates, including ethanol, 1‐propanol, 2‐propanol, butanol, acetaldehyde, propanal, and acetone, were 4.36 ± 0.42, 4.69 ± 0.41, 3.74 ± 0.46, 2.44 ± 0.30, 1.27 ± 0.18, 1.55 ± 0.20, and 0.68 ± 0.04 mM, respectively. The optimal pH values for catalyzing aldehyde reduction and alcohol oxidation were 6.0 and 9.0, respectively, while the optimal temperature was higher than 90°C due to the increase in enzymatic activity from 60°C to 90°C. Based on its substrate specificity, enzyme kinetics, and thermostability, HbADH2 may be the ADH that catalyzes the production of 1‐butanol in H. butylicus. The putative conserved motif sites for NAD(P)+ and iron binding were identified by aligning HbADH2 with previously characterized Fe‐containing ADHs.

Publisher

Wiley

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