title: 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.
abstract: 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 alpha (54 kDa), beta (43 kDa), gamma (19 kDa), and delta (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 alphabetagammadelta structure. Functional analysis of each subunit revealed that the beta subunit catalyzed the dye-linked l-proline dehydrogenase reaction by itself and that, unexpectedly, the alpha 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.
authors.affiliation: Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, 2-1 Minamijosanjimacho, Tokushima 770-8506, Japan.
authors.forename: Ryushi
authors.initials: R
authors.lastname: Kawakami
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Sakuraba
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohshima
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004396:Coloring Agents / Q000737:chemistry*; D006358:Hot Temperature; D046911:Macromolecular Substances / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family / Q000235:genetics; D011394:Proline Oxidase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics*
chemical_list: D004396:Coloring Agents; D046911:Macromolecular Substances; D021122:Protein Subunits; D011394:Proline Oxidase