Purification and characterization of 2-oxoglutarate: Ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6

K. I.Seok Yoon, Masaharu Ishii, Yasuo Igarashi, Tohru Kodama

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK- 6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80°C, and the time for a 50% loss of activity at 70°C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent K(m) values for 2-oxoglutarate and coenzyme A at 70°C were 1.42 mM and 80 μM, respectively.

Original languageEnglish
Pages (from-to)3365-3368
Number of pages4
JournalJournal of bacteriology
Volume178
Issue number11
DOIs
Publication statusPublished - Jun 1996
Externally publishedYes

    Fingerprint

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

Cite this