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Production and characterization of a thermostable L ‐threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus
Author(s) -
Machielsen Ronnie,
van der Oost John
Publication year - 2006
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05290.x
Subject(s) - pyrococcus furiosus , threonine , thermostability , biochemistry , hyperthermophile , chemistry , enzyme , serine , nad+ kinase , escherichia coli , stereochemistry , biology , gene , archaea
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 Zn 2+ and Co 2+ for activity and contains at least one zinc atom per subunit. K m values for l ‐threonine and NAD + at 70 °C were 1.5 m m and 0.055 m m , respectively.