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

M.P. Machielsen, J. van der Oost

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17 Citations (Scopus)

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 ¿ 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 Co 2+ 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
Original languageEnglish
Pages (from-to)2722-2729
JournalFEBS Journal
Volume273
Issue number12
DOIs
Publication statusPublished - 2006

Keywords

  • containing alcohol-dehydrogenase
  • site-directed mutagenesis
  • escherichia-coli
  • horikoshii
  • proteins
  • binding
  • identification
  • histidine-90
  • degradation
  • catabolism

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