Alcohol dehydrogenases (ADHs) are enzymes that catalyze redox reactions between alcohols and their corresponding aldehydes and ketones. NAD(P)-dependent ADHs, which account for the majority of ADHs, are classified into group I, II, and III based on the homology of their amino acid sequences. We have been studying group III ADH (PhADH) derived from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. The N-terminal domain (N-domain), corresponding to the amino acid sequence Met1-Asn182, binds to the coenzyme NAD(H). On the other hand, the C-terminal domain (Met183-Asp375) (C-domain) contains the metal ion and amino acid residues required for catalytic activity. We focused on the domain structure of PhADH and hypothesized that if enzymatic activity was confirmed when PhADH was cut into two domains and mixed in solution, it might be possible to construct a new system in solution or electrode systems that differs from the full-length PhADH. In this study, we genetically fragmented PhADH into two domains, investigated the properties of each domain and the enzymatic activity in a domain-mixed system, and compared them with the full-length PhADH reported in our previous study. (1)
The amount of dissolved protein remained high up to 60°C for the N-domain and 70°C for the C-domain. This indicates that each domain has relatively high thermal stability. Although each domain alone obviously does not show enzymatic activity, a mixture of the two domains was found to show activity close to that of the full-length enzyme. The substrate and pH dependence of the aldehyde reduction activity of the domain mixture was examined. The substrate and pH dependence of the domain mixture for the aldehyde reduction reaction showed similar trends to those of the full-length enzyme, but differences were observed in the reactivity to substrates other than the optimal substrate and in the optimal pH. These results suggested that 1) when the two domains are mixed in solution, they form an aggregate that exhibits enzymatic activity, and 2) the structure near the active center of this aggregate is slightly different from that of the full-length enzyme, resulting in a slight change in reactivity.