, Volume 163, Issue 4, pp 286-290

Purification and characterization of threonine dehydrogenase from Clostridium sticklandii

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Abstract

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-threo-nine, dl-β-hydroxynorvaline and acetoin were substrates; only NAD was used as the natural electron acceptor. The apparent K m 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.