Gene and primary structures of dye-linked l-proline dehydrogenase from the hyperthermophilic archaeon Thermococcus profundus show the presence of a novel heterotetrameric amino acid dehydrogenase complex
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Dye-linked l-proline dehydrogenase catalyzes the oxidation of l-proline in the presence of artificial electron acceptors such as 2, 6-dichloroindophenol and ferricyanide. The enzyme from the hyperthermophilic archaeon Thermococcus profundus was purified and characterized for the first time in archaea by Sakuraba et al. in 2001. In this study, cloning and sequencing analyses of the gene encoding the enzyme and functional analysis of the subunits were performed. The gene formed an operon that consisted of four genes, pdhA, pdhB, pdhF, and pdhX, which are tandemly arranged in the order of pdhA-F-X-B. SDS-PAGE analysis of the purified recombinant enzyme showed four different bands corresponding to α (54 kDa), β (43 kDa), γ (19 kDa), and δ (8 kDa) subunits encoded by pdhA, pdhB, pdhF, and pdhX, respectively, and the molecular ratio of these subunits was determined to be equal. This indicates that the enzyme consists of a heterotetrameric αβγδ structure. Functional analysis of each subunit revealed that the β subunit catalyzed the dye-linked l-proline dehydrogenase reaction by itself and that, unexpectedly, the α subunit exhibited dye-linked NADH dehydrogenase activity. This is the first example showing the existence of a bifunctional dye-linked l-proline/NADH dehydrogenase complex. On the basis of genome analysis, similar gene clusters were observed in the genomes of Pyrococcus horikoshii, Pyrococcus abyssi, Pyrococcus furiosus, and Archaeoglobus fulgidus. These results indicate that the dye-linked l-proline dehydrogenase is a novel type of heterotetrameric amino acid dehydrogenase that might be widely distributed in the hyperthermophilic archaeal strain.
KeywordsBifunctional enzyme Dye-linked l-proline dehydrogenase Dye-linked NADH dehydrogenase Heterotetrameric structure Hyperthermophilic archaea Thermococcus profundus
This study was supported by the Pioneering Research Project in Biotechnology of the Ministry of Agriculture, Forestry and Fisheries. R.K. was partially supported by the Sasakawa Scientific Research Grant from The Japan Science Society.
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