Purification and characterization of pyruvate:ferredoxin oxidoreductase from hydrogenobacter thermophilus TK-6

Ki Seok Yoon, Masaharu Ishii, Tohru Kodama, Yasuo Igarashi

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Abstract

Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogenoxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-GB, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an αβγδ-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron accepters in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, ana ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80°C. The pH optimum was 7.6-7.8. The apparent K(m) values for pyruvate and coenzyme A at 70°C were 3.45 mM and 54 μM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t(50%)) at 70°C was approximately 8 h.

Original languageEnglish
Pages (from-to)275-279
Number of pages5
JournalArchives of Microbiology
Volume167
Issue number5
DOIs
Publication statusPublished - Apr 28 1997
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Biochemistry
  • Molecular Biology
  • Genetics

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