Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea

被引:97
作者
Heider, J
Mai, XH
Adams, MWW
机构
[1] UNIV GEORGIA,DEPT BIOCHEM & MOLEC BIOL,ATHENS,GA 30602
[2] UNIV GEORGIA,CTR METALLOENZYME STUDIES,ATHENS,GA 30602
关键词
D O I
10.1128/jb.178.3.780-787.1996
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 (k(cat)/K-m > 1.0 mu M(-1) s(-1); K-m < 100 mu M) for the enzyme were the 2-ketoacid derivatives of valine, leucine, isoleucine, and methionine, while pyruvate and aryl pyruvates were very poor substrates (k(cat)/K-m < 0.2 mu M(-1) s(-1)) and 2-ketoglutarate was not utilized. T. litoralis VOR also functioned as a 2-keto-isovalerate synthase at 85 degrees C, producing 2-ketoisovalerate and coenzyme A from isobutyryl-coenzyme A (apparent K-m, 250 mu M) and CO2 (apparent K-m, 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.
引用
收藏
页码:780 / 787
页数:8
相关论文
共 68 条