Purification and characterization of threonine dehydrogenase from Clostridium sticklandii.
Threonine dehydrogenase from Clostridium sticklandii has been purified 76-fold from cells grown in a defined medium to a homogeneous preparation of 234 units.mg-1 protein. Purification was obtained by chromatography on Q-Sepharose fast flow and Reactive green 19-Agarose. The native enzyme had a molecular mass of 67 kDa and consisted of two identical subunits (33 kDa each). The optimum pH for catalytic activity was 9.0. Only L-threo-threonine, DL-beta-hydroxynorvaline and acetoin were substrates; only NAD was used as the natural electron acceptor. The apparent Km values for L-threonine and NAD were 18 mM and 0.1 mM, respectively. Zn2+, Co2+ and Cu2+ ions (0.9 mM) inhibited enzyme activity. The N-terminal amino acid sequence revealed similarities to the class of non-metal short-chain alcohol dehydrogenases, whereas the threonine dehydrogenase from Escherichia coli belongs to the class of medium chain, zinc-containing alcohol dehydrogenases.
Wagner
M
M
Institut für Mikrobiologie, Universität Göttingen, Germany.
Andreesen
J R
JR
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D003013:Clostridium / Q000201:enzymology*; D008969:Molecular Sequence Data
D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase