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FEBS J. 2006 Jun;273(12):2722-9.

Production and characterization of a thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

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1
Laboratory of Microbiology, Wageningen University, the Netherlands. Ronnie.machielsen@wur.nl

Abstract

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.

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