Production and characterization of a thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.
The gene encoding a threonine dehydrogenase (TDH) has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The Pf-TDH protein has been functionally produced in Escherichia coli and purified to homogeneity. The enzyme has a tetrameric conformation with a molecular mass of approximately 155 kDa. The catalytic activity of the enzyme increases up to 100 degrees C, and a half-life of 11 min at this temperature indicates its thermostability. The enzyme is specific for NAD(H), and maximal specific activities were detected with L-threonine (10.3 U x mg(-1)) and acetoin (3.9 U x mg(-1)) in the oxidative and reductive reactions, respectively. Pf-TDH also utilizes L-serine and D-threonine as substrate, but could not oxidize other L-amino acids. The enzyme requires bivalent cations such as Zn2+ and Co2+ for activity and contains at least one zinc atom per subunit. Km values for L-threonine and NAD+ at 70 degrees C were 1.5 mm and 0.055 mm, respectively.
Machielsen
Ronnie
R
Laboratory of Microbiology, Wageningen University, the Netherlands. Ronnie.machielsen@wur.nl
van der Oost
John
J
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D020027:Pyrococcus furiosus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D013722:Teprotide / Q000378:metabolism / Q000494:pharmacology
D002413:Cations, Divalent; D003067:Coenzymes; D011994:Recombinant Proteins; D013722:Teprotide; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase