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摘要
The activity of dye-linked D-proline dehydrogenase was found in the crude extract of a hyperthermophilic archacon, 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 MKVAIVGGGIIGLFTAYHL-RQQGADVVI. The enzyme retained its full activity both after incubation at 80 degreesC for 10 min and after incubation in the range of pH 4.0-10.0 at 50 degreesC 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.
- 出版日期2002-4-12
