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 ≈ 155 kDa. The catalytic activity of the enzyme increases up to 100 °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·mg−1) and acetoin (3.9 U·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 °C were 1.5 mm and 0.055 mm, respectively.