Archives of Microbiology

, Volume 163, Issue 4, pp 286–290

Purification and characterization of threonine dehydrogenase from Clostridium sticklandii

  • Matthias Wagner
  • Jan R. Andreesen
Original Paper

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 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.

Key words

Clostridium sticklandii Threonine dehydrogenase Aminoacetone Alcohol dehydrogenase 

Abbreviations

PMSF

phenylmethylsulfonyl fluoride

Dea

diethanolamine

Tris

tris-(hydroxy-methyl)-aminomethane

Nbs2

5,5′-dithiobis-(2-nitrobenzoic acid)

ApADN

3-acetylpyridine adenine diucleotide

thio-NAD

thionicotinamide adenine dinucleotide

NBT

nitro blue tetrazolium chloride

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Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Matthias Wagner
    • 1
  • Jan R. Andreesen
    • 1
  1. 1.Institut für MikrobiologieUniversität GöttingenGöttingenGermany
  2. 2.Institut für MkrobiologieUniversität HalleHalle/SaaleGermany

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