The Catalytic Mechanism of 2‐Oxoacid: Ferredoxin Oxidoreductases from <i>Halobacterium halobium</i>

Kerscher, Lorenz; Oesterhelt, Dieter

doi:10.1111/j.1432-1033.1981.tb05377.x

Cited by 92 publications

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References 21 publications

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“…From the rhombic-type line shape of the spectrum, this originates predominately from reduced FeS-1. The first step of the mechanism of T. litoralis VOR therefore proceeds in a manner analogous to that proposed for the POR from Halobacterium halobium, with the formation of an initial acyl-TPP adduct and the immediate abstraction of one electron by an adjacent FeS cluster (FeS-1) to give a hydroxyacyl-TPP radical (31). By analogy with P. furiosus VOR, it seems likely that FeS-1 is the cluster which is coordinated by the TPP-containing ␤ subunit of T. litoralis VOR.…”

Section: S]mentioning

confidence: 99%

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

1996

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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 (␣), 34,000 (␤), 23,000 (␥), and 13,000 (␦). It contains about 11 iron and 12 acid-labile sulfide atoms and 13 cysteine residues per heterotetramer (␣␤␥␦), but thiamine pyrophosphate, which is required for catalytic activity, was lost during purification. The most efficient substrates (k cat /K m > 1.0 M ؊1 s ؊1; K m < 100 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 M ؊1 s ؊1) and 2-ketoglutarate was not utilized. T. litoralis VOR also functioned as a 2-ketoisovalerate synthase at 85؇C, producing 2-ketoisovalerate and coenzyme A from isobutyryl-coenzyme A (apparent K m , 250 M) and CO 2 (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.In the last decade, a remarkable group of microorganisms capable of growing at temperatures around 100ЊC have been isolated from geothermally heated habitats (59,60). Virtually all of these so-called hyperthermophiles are classified as archaea (formerly archaebacteria [68]). Most of them are obligately anaerobic organisms and obtain energy for growth by either fermentation, sulfate reduction, or methanogenesis (1, 2, 28). The fermentative hyperthermophiles typically require elemental sulfur (S 0 ), which is reduced to H 2 S, for growth, although some, such as species of Pyrococcus and Thermococcus, grow well in the absence of S 0 (22,38,48). A characteristic of these organisms is their ability to ferment peptides, and some can also utilize carbohydrates (28,59,60). The pathways of carbohydrate fermentation have recently been shown to resemble the well-known bacterial pathways but with some unique additions and possibly a combination of different pathways (29,46,56).Only limited information is available on the degradation of amino acids by hyperthermophilic archaea (2, 28). Several of these organisms have been shown to contain high protease (9, 21, 33), glutamate dehydrogenase (16,19,40,54) and aromatic amino acid transaminase (3, 4) activities, and these enzymes have been purified from one or more species. In addition, three ...

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Section: S]mentioning

confidence: 99%

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

1996

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“…1b). Instead, the hydroxyethyl group is transferred directly from thiamine pyrophosphate (TPP) to Coenzyme-A and the reducing equivalents are passed via an iron-sulphur centre to ferredoxin (Kerscher et al, 1981).…”

Section: Introductionmentioning

confidence: 99%

2-Oxoacid dehydrogenase multienzyme complexes in the halophilic Archaea? Gene sequences and protein structural predictions The GenBank accession number for the sequence reported in this paper is AF068743.

et al. 2000

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All Archaea catalyse the conversion of pyruvate to acetyl-CoA via a simple pyruvate oxidoreductase. This is in contrast to the Eukarya and most aerobic bacteria, which use the pyruvate dehydrogenase multienzyme complex [PDHC], consisting of multiple copies of three component enzymes : E1 (pyruvate decarboxylase), E2 (lipoate acetyl-transferase) and E3 (dihydrolipoamide dehydrogenase, DHLipDH). Until now no PDHC activity has been found in the Archaea, although DHLipDH has been discovered in the extremely halophilic Archaea and its gene sequence has been determined. In this paper, the discovery and sequencing of an operon containing the DHLipDH gene in the halophilic archaeon Haloferax volcanii are reported. Upstream of the DHLipDH gene are 3 ORFs which show highest sequence identities with the E1α, E1β and E2 genes of the PDHC from Gram-positive organisms. Structural predictions of the proposed protein product of the E2 gene show a domain structure characteristic of the E2 component in PDHCs, and catalytically important residues, including the lysine to which the lipoic acid cofactor is covalently bound, are conserved. Northern analyses indicate the transcription of the whole operon, but no PDHC enzymic activity could be detected in cell extracts. The presence in the E2 gene of an insertion (equivalent to approximately 100 aa) not found in bacterial or eukaryal E2 proteins, might be predicted to prevent multienzyme complex assembly. This is the first detailed report of the genes for a putative 2-oxoacid dehydrogenase complex in the Archaea, and the evolutionary and metabolic consequences of these findings are discussed.

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“…VOR and KGOR also contain four distinct subunits with sizes comparable to those of the PORs, whereas IOR consists only of two subunits (A and B) with sizes of 66 and 23 kDa (17,31,32). As with POR, the acyl-and aryl-CoA derivatives produced by the three other ORs are thought to be utilized for energy conservation (1a, 2, 22).PORs have also been purified from various mesophilic organisms, including several anaerobic bacteria (8,20,33,37,44,45), aerobic archaeal halobacteria (23,24,38), and anaerobic eucaryotes (10, 47). On the other hand, in most aerobic organisms, pyruvate oxidation is catalyzed by a pyruvate dehydrogenase (PDH) complex (reviewed in reference 46).…”

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“…PORs have also been purified from various mesophilic organisms, including several anaerobic bacteria (8,20,33,37,44,45), aerobic archaeal halobacteria (23,24,38), and anaerobic eucaryotes (10, 47). On the other hand, in most aerobic organisms, pyruvate oxidation is catalyzed by a pyruvate dehydrogenase (PDH) complex (reviewed in reference 46).…”

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

1996

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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 PP i -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 ␥ 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 ␦ subunits each contained two ferredoxin-type [4Fe-4S] cluster binding motifs (CXXCXXC XXXCP), while their ␤ subunits each contained four conserved cysteines in addition to a thiamine PP i -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.The final oxidative step in the fermentation of carbohydrates in anaerobic microorganisms is typically catalyzed by pyruvate ferredoxin oxidoreductase (POR). This involves the oxidative decarboxylation of pyruvate with the participation of thiamine PP i (TPP) followed by the transfer of an acetyl moiety to coenzyme A (CoASH) for the synthesis of acetyl-CoA as shown in the following equation: CH 3 -CO-COOH ϩ CoASH 3 CH 3 -CO-SCoA ϩ CO 2 ϩ 2H ϩ ϩ 2e Ϫ (e Ϫ is an electron) (44, 45). The electrons from this reaction are transferred to low-potential ferredoxins or flavodoxins typically for ultimate disposal either as H 2 or in an organic compound.PORs have been purified from several anaerobic hyperthermophiles, which are organisms that grow at temperatures near and above 100ЊC (42). These include the sulfur-reducing archaea Pyrococcus furiosus (6) and Thermococcus litoralis (15), the sulfate-reducing archaeon Archaeoglobus fulgidus (28), and the bacterium Thermotoga maritima (7). All the hyperthermophilic PORs comprise four different subunits (␣, ␤, ␥, and ␦) with apparent molecular masses of approximately 43, 35, 23, and 12 kDa, respectively. We have also purified three other types ...

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The Catalytic Mechanism of 2‐Oxoacid: Ferredoxin Oxidoreductases from Halobacterium halobium

Cited by 92 publications

References 21 publications

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

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

2-Oxoacid dehydrogenase multienzyme complexes in the halophilic Archaea? Gene sequences and protein structural predictions The GenBank accession number for the sequence reported in this paper is AF068743.

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

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