title: A method of expression for an oxygen-tolerant group III alcohol dehydrogenase from Pyrococcus horikoshii OT3.
abstract: NAD(P)-dependent group III alcohol dehydrogenases (ADHs), well known as iron-activated enzymes, generally lose their activities under aerobic conditions due to their oxygen-sensitivities. In this paper, we expressed an extremely thermostable group III ADH from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (PhADH) heterologously in Escherichia coli. When purified from a culture medium containing nickel, the recombinant PhADH (Ni-PhADH) contained 0.85 ± 0.01 g-atoms of nickel per subunit. Ni-PhADH retained high activity under aerobic conditions (9.80 U mg-1), while the enzyme expressed without adding nickel contained 0.46 ± 0.01 g-atoms of iron per subunit and showed little activity (0.27 U mg-1). In the presence of oxygen, the activity of the Fe2+-reconstituted PhADH prepared from the Ni-PhADH was gradually decreased, whereas the Ni2+-reconstituted PhADH maintained enzymatic activity. These results indicated that PhADH with bound nickel ion was stable in oxygen. The activity of the Ni2+-reconstituted PhADH prepared from the expression without adding nickel was significantly lower than that from the Ni-PhADH, suggesting that binding a nickel ion to PhADH in this expression system contributed to protecting against inactivation during the expression and purification processes. Unlike other thermophilic group III ADHs, Ni-PhADH showed high affinity for NAD(H) rather than NADP(H). Furthermore, it showed an unusually high kcatvalue toward aldehyde reduction. The activity of Ni-PhADH for butanal reduction was increased to 60.7 U mg-1with increasing the temperature to 95 °C. These findings provide a new strategy to obtain oxygen-sensitive group III ADHs.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo, 184-8588, Japan.
authors.forename: Chikanobu
authors.initials: C
authors.lastname: Sugimoto
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo, 184-8588, Japan.
authors.forename: Kouta
authors.initials: K
authors.lastname: Takeda
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo, 184-8588, Japan.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Kariya
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita, 010-8502, Japan.
authors.forename: Hirotoshi
authors.initials: H
authors.lastname: Matsumura
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo, 184-8588, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yohda
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo, 184-8588, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Ohno
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo, 184-8588, Japan. nobu1@cc.tuat.ac.jp.
authors.forename: Nobuhumi
authors.initials: N
authors.lastname: Nakamura
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D010100:Oxygen / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D013696:Temperature
chemical_list: D000426:Alcohol Dehydrogenase; D010100:Oxygen
keywords: Alcohol dehydrogenase (ADH); Archaea; Extreme thermophile; Metalloenzyme; Nickel