Abstract: An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40±1 kDa. The gene encoding the enzyme was cloned and sequenced, and found to have significant sequence homology to known zinc-containing ADHs and L-threonine dehydrogenases with both binding motifs of catalytic zinc and NADP+. The wild-type enzyme is a primary-secondary ADH that exhibits a substrate preference for secondary alcohols and corresponding ketones, and exhibits unusual stereoselectivity. The wild-type enzyme was found to have outstanding thermostability, demonstrating 60% activity after incubation at 80° C. for 40 hours. Site-directed mutagenesis was used to substitute the cyteine residue at position 56 with a serine, to provide the TgADH(C56S) mutant. In the assays that we carried out, we found virtually no difference in enzyme activity and oxygen-sensitivity between the mutant TgADH (C56S) and wild type TgADH.

Abstract: An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40±1 kDa. The gene encoding the enzyme was cloned and sequenced, and found to have significant sequence homology to known zinc-containing ADHs and L-threonine dehydrogenases with both binding motifs of catalytic zinc and NADP+. The wild-type enzyme is a primary-secondary ADH that exhibits a substrate preference for secondary alcohols and corresponding ketones, and exhibits unusual stereoselectivity. The wild-type enzyme was found to have outstanding thermostability, demonstrating 60% activity after incubation at 80° C. for 40 hours. Site-directed mutagenesis was used to substitute the cyteine residue at position 56 with a serine, to provide the TgADH(C56S) mutant. In the assays that we carried out, we found virtually no difference in enzyme activity and oxygen-sensitivity between the mutant TgADH (C56S) and wild type TgADH.