title: Dye-linked D-proline dehydrogenase from hyperthermophilic archaeon Pyrobaculum islandicum is a novel FAD-dependent amino acid dehydrogenase.
abstract: The activity of dye-linked d-proline dehydrogenase was found in the crude extract of a hyperthermophilic archaeon, Pyrobaculum islandicum JCM 9189. The dye-linked d-proline dehydrogenase was a membrane associated enzyme and was solubilized from the membrane fractions by treatment with Tween 20. The solubilized enzyme was purified 34-fold in the presence of 0.1% Tween 20 by four sequential chromatographies. The enzyme has a molecular mass of about 145 kDa and consisted of homotetrameric subunits with a molecular mass of about 42 kDa. The N-terminal amino acid sequence of the subunit was MKVAIVGGGIIGLFTAYHLRQQGADVVI. The enzyme retained its full activity both after incubation at 80 degrees C for 10 min and after incubation in the range of pH 4.0-10.0 at 50 degrees C for 10 min. The enzyme-catalyzed dehydrogenation of several d-amino acids was carried out using 2,6-dichloroindophenol as an electron acceptor, and d-proline was the most preferred substrate among the d-amino acids. The Michaelis constants for d-proline and 2,6-dichloroindophenol were determined to be 4.2 and 0.14 mm, respectively. Delta(1)-Pyrroline-2-carboxylate was identified as the reaction product from d-proline by thin layer chromatography. The prosthetic group of the enzyme was identified to be FAD by high-performance liquid chromatography. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the dye-linked d-proline dehydrogenase gene was determined and encoded a peptide of 363 amino acids with a calculated molecular weight of 40,341. The amino acid sequence of the Pb. islandicum enzyme showed the highest similarity (38%) with that of the probable oxidoreductase in Sulfolobus solfataricus, but low similarity with those of d-alanine dehydrogenases from the mesophiles so far reported. This shows that the membrane-bound d-proline dehydrogenase from Pb. islandicum is a novel FAD-dependent amino acid dehydrogenase.
authors.affiliation: Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minami-josanjimacho, Tokushima 770-8506, Japan.
authors.forename: Takenori
authors.initials: T
authors.lastname: Satomura
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; D002852:Chromatography, Ion Exchange; D004396:Coloring Agents / Q000737:chemistry*; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D011394:Proline Oxidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019624:Thermoproteaceae / Q000201:enzymology*
chemical_list: D004396:Coloring Agents; D015342:DNA Probes; D011394:Proline Oxidase