Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea.
Cell extracts of the proteolytic and hyperthermophilic archaea Thermococcus litoralis, Thermococcus sp. strain ES-1, Pyrococcus furiosus, and Pyrococcus sp. strain ES-4 contain an enzyme which catalyzes the coenzyme A-dependent oxidation of branched-chain 2-ketoacids coupled to the reduction of viologen dyes or ferredoxin. This enzyme, termed VOR (for keto-valine-ferredoxin oxidoreductase), has been purified from all four organisms. All four VORs comprise four different subunits and show amino-terminal sequence homology. T. litoralis VOR has an M(r) of ca. 230,000, with subunit M(r) values of 47,000 (alpha), 34,000 (beta), 23,000 (gamma), and 13,000 (delta). It contains about 11 iron and 12 acid-labile sulfide atoms and 13 cysteine residues per heterotetramer (alpha beta gamma delta), but thiamine pyrophosphate, which is required for catalytic activity, was lost during purification. The most efficient substrates (kcat/Km > 1.0 microM-1 s-1; Km < 100 microM) for the enzyme were the 2-ketoacid derivatives of valine, leucine, isoleucine, and methionine, while pyruvate and aryl pyruvates were very poor substrates (kcat/Km < 0.2 microM-1 s-1) and 2-ketoglutarate was not utilized. T. litoralis VOR also functioned as a 2-ketoisovalerate synthase at 85 degrees C, producing 2-ketoisovalerate and coenzyme A from isobutyryl-coenzyme A (apparent Km, 250 microM) and CO2 (apparent Km, 48 mM) with reduced viologen as the electron donor. The rate of 2-ketoisovalerate synthesis was about 5% of the rate of 2-ketoisovalerate oxidation. The optimum pH for both reactions was 7.0. A mechanism for 2-ketoisovalerate oxidation based on data from substrate-induced electron paramagnetic resonance spectra is proposed, and the physiological role of VOR is discussed.
Heider
J
J
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602, USA.
Mai
X
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Adams
M W
MW
D000595:Amino Acid Sequence; D001105:Archaea / Q000378:metabolism*; D003065:Coenzyme A / Q000502:physiology*; D004578:Electron Spin Resonance Spectroscopy; D005285:Fermentation; D045782:Hemiterpenes; D007651:Keto Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*
D045782:Hemiterpenes; D007651:Keto Acids; D010455:Peptides; C001505:alpha-ketoisovalerate; D010088:Oxidoreductases; D003065:Coenzyme A