Publications

• Molecular and phylogenetic characterization of pyruvate and 2-ketoisovalerate ferredoxin oxidoreductases from Pyrococcus furiosus and pyruvate ferredoxin oxidoreductase from Thermotoga maritima.

  Kletzin A., Adams M.W.W.

  Previous studies have shown that the hyperthermophilic archaeon Pyrococcus furiosus contains four distinct cytoplasmic 2-ketoacid oxidoreductases (ORs) which differ in their substrate specificities, while the hyperthermophilic bacterium Thermotoga maritima contains only one, pyruvate ferredoxin ox ... >> More

  Previous studies have shown that the hyperthermophilic archaeon Pyrococcus furiosus contains four distinct cytoplasmic 2-ketoacid oxidoreductases (ORs) which differ in their substrate specificities, while the hyperthermophilic bacterium Thermotoga maritima contains only one, pyruvate ferredoxin oxidoreductase (POR). These enzymes catalyze the synthesis of the acyl (or aryl) coenzyme A derivative in a thiamine PPi-dependent oxidative decarboxylation reaction with reduction of ferredoxin. We report here on the molecular analysis of the POR (por) and 2-ketoisovalerate ferredoxin oxidoreductase (vor) genes from P. furiosus and of the POR gene from T. maritima, all of which comprise four different subunits. The operon organization for P. furiosus POR and VOR was porG-vorDAB-porDAB, wherein the gamma subunit is shared by the two enzymes. The operon organization for T. maritima POR was porGDAB. The three enzymes were 46 to 53% identical at the amino acid level. Their delta subunits each contained two ferredoxin-type [4Fe-4S] cluster binding motifs (CXXCXXCXXXCP), while their beta subunits each contained four conserved cysteines in addition to a thiamine PPi-binding domain. Amino-terminal sequence comparisons show that POR, VOR, indolepyruvate OR, and 2-ketoglutarate OR of P. furiosus all belong to a phylogenetically homologous OR family. Moreover, the single-subunit pyruvate ORs from mesophilic and moderately thermophilic bacteria and from an amitochondriate eucaryote each contain four domains which are phylogenetically homologous to the four subunits of the hyperthermophilic ORs (27% sequence identity). Three of these subunits are also homologous to the dimeric POR from a mesophilic archaeon, Halobacterium halobium (21% identity). A model is proposed to account for the observed phenotypes based on genomic rearrangements of four ancestral OR subunits. << Less

  J. Bacteriol. 178:248-257(1996) [PubMed] [EuropePMC]

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

  Heider J., Mai X., Adams M.W.W.

  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 vi ... >> More

  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. << Less

  J. Bacteriol. 178:780-787(1996) [PubMed] [EuropePMC]

• Structures and functions of four anabolic 2-oxoacid oxidoreductases in Methanobacterium thermoautotrophicum.

  Tersteegen A., Linder D., Thauer R.K., Hedderich R.

  Methanobacterium thermoautotrophicum (strain Marburg), which grows autotrophically on H2 and CO2, was found to contain 2-oxoisovalerate oxidoreductase (Vor) and indolepyruvate oxidoreductase (Ior) besides pyruvate oxidoreductase (Por) and 2-oxoglutarate oxidoreductase (Kor). So far, Vor and Ior ha ... >> More

  Methanobacterium thermoautotrophicum (strain Marburg), which grows autotrophically on H2 and CO2, was found to contain 2-oxoisovalerate oxidoreductase (Vor) and indolepyruvate oxidoreductase (Ior) besides pyruvate oxidoreductase (Por) and 2-oxoglutarate oxidoreductase (Kor). So far, Vor and Ior have only been detected in peptide-utilizing hyperthermophilic Archaea. The four 2-oxoacid oxidoreductases were purified and characterized with respect to their subunit composition, N-terminal amino acid sequences, and catalytic properties. Por and Kor were composed of four different subunits, Vor was composed of three different subunits, and Ior of two different subunits. Comparisons of the N-terminal amino acid sequences revealed that the four enzymes are structurally related to each other and to the respective enzymes from Pyrococcus and Thermococcus sp. Vor from M. thermoautotrophicum differed from Vor from Pyrococcus furiosus in being composed of only three instead of four different subunits. Evidence is presented that in the autotrophic methanogen the four 2-oxoacid oxidoreductases have anabolic functions, Vor and Ior being involved in the biosynthesis of amino acids from fatty acids taken up from the growth medium, as shown by 14C-labelling studies. << Less

  Eur. J. Biochem. 244:862-868(1997) [PubMed] [EuropePMC]

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