title: Dye-linked D-amino acid dehydrogenase from the thermophilic bacterium Rhodothermus marinus JCM9785: characteristics and role in trans-4-hydroxy-L-proline catabolism.
abstract: A gene from the thermophilic Gram-negative bacterium Rhodothermus marinus JCM9785, encoding a dye-linked D-amino acid dehydrogenase homologue, was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was a highly thermostable dye-linked D-amino acid dehydrogenase that retained more than 80% of its activity after incubation for 10 min at up to 70 °C. When enzyme-catalyzed dehydrogenation of several D-amino acids was carried out using 2,6-dichloroindophenol as the electron acceptor, D-phenylalanine was the most preferable substrate among the D-amino acids tested. Immediately upstream of the dye-linked D-amino acid dehydrogenase gene (dadh) was a gene encoding a 4-hydroxyproline 2-epimerase homologue (hypE). That gene was successfully expressed in E. coli, and the gene product exhibited strong 4-hydroxyproline 2-epimerase activity. Reverse transcription PCR and quantitative real-time PCR showed that the six genes containing the dadh and hypE genes were arranged in an operon and were required for catabolism of trans-4-hydroxy-L-proline in R. marinus. This is the first description of a dye-linked D-amino acid dehydrogenase (Dye-DADH) with broad substrate specificity involved in trans-4-hydroxy-L-proline catabolism.
authors.affiliation: Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui, 910-8507, Japan, satomura@u-fukui.ac.jp.
authors.forename: Takenori
authors.initials: T
authors.lastname: Satomura
authors.forename: Masaru
authors.initials: M
authors.lastname: Ishikura
authors.forename: Takashi
authors.initials: T
authors.lastname: Koyanagi
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Sakuraba
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohshima
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Suye
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D011392:Proline / Q000737:chemistry / Q000378:metabolism*; D045245:Rhodothermus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011392:Proline; D000594:Amino Acid Oxidoreductases