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.

L-Proline sensor based on layer-by-layer immobilization of thermostable dye-linked L-proline dehydrogenase and polymerized mediator

“…The general properties of this thermostable dye-linked L-proDH were thoroughly characterized by Sakuraba et al [11,12]. The enzyme (molecular weight 160 kDa) consists of four subunits (abgd), and b subunit catalyzes the dye-linked L-proline dehydrogenase reaction.…”

“…The activity of the enzyme was routinely determined spectrophotometrically by measuring the reduction rate of DCPIP at 595 nm according to the previous procedures described [11,12]. The reaction mixture was composed of 200 mM Tris-HCl buffer (pH 7.5), 0.1 mM DCPIP, 100 mM L-proline and enzyme solution with a total volume of 1.0 ml.…”

“…Previously, dye-linked L-proline dehydrogenase (L-proDH) originally from hyperthermophilic archaeon (Thermococcus profundus) has been expressed as a recombinant protein [11,12]. The enzyme showed high thermal stability, and a dye compound such as 2,6-dichlorophenolindophenol (DCPIP) could be used as an artificial electron acceptor for enzyme reaction.…”

Crystal Structure of Novel Dye-linked l-Proline Dehydrogenase from Hyperthermophilic Archaeon Aeropyrum pernix

“…Two different types of LPDH, PDH1 and PDH2, have been identified in the anaerobic hyperthermophile Pyrococcus horikoshii OT-3 (7). PDH1 is a hetero-octameric complex (␣4␤4; molecular mass, 440 kDa) that also contains FAD, FMN, Fe 3ϩ , and ATP, whereas PDH2 is a heterotetrameric complex (␣␤␥␦; molecular mass, 120 kDa) composed of an L-proline dehydrogenase, an NADH dehydrogenase, a ferredoxin-like protein, and a protein of unknown function (6,7). Using x-ray crystallography, we solved the three-dimensional structure of the PDH1 complex (11), which we found to be a unique diflavin dehydrogenase containing a novel electron transfer system that is totally different from that of the PDH2 complex.…”

“…Using x-ray crystallography, we solved the three-dimensional structure of the PDH1 complex (11), which we found to be a unique diflavin dehydrogenase containing a novel electron transfer system that is totally different from that of the PDH2 complex. Proteins homologous to PDH1 and PDH2 are widely distributed among the anaerobic hyperthermophilic archaea that belong to the phylum Euryarchaeota (6,7).…”