Characterization of aromatic aminotransferases from the hyperthermophilic archaeon <i>Thermococcus litoralis</i>

Andreotti, Giuseppina; Cubellis, Maria Vittoria; Nitti, Gianpaolo; Sannia, Giovanni; Mai, Xuhong; Marino, Gennaro; Adams, Michael W. W.

doi:10.1111/j.1432-1033.1994.tb18654.x

Cited by 36 publications

(26 citation statements)

References 42 publications

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“…litoralis is a heterotrophic organism which was initially described as growing on peptides and pyruvate but not on carbohydrates (22). Several enzymes supposedly involved in peptide metabolism in this organism have been described (1,14,17,20,24). In contrast, very little is known about carbohydrate metabolism.…”

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High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis

et al. 1996

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The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85؇C. The K m for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the V max for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14 C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 M concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, K i , of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [ 14 C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 M maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M r of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.

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High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis

et al. 1996

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“…In addition to pyruvate oxidation, peptides and amino acids also are dissimilated simultaneously (11). Amino acids, which are directly incorporated into the cells or formed by degradation of peptides, are deaminated to the corresponding 2-oxoacids using 2-oxoglutarate as an amino acceptor by a number of aminotransferases with different substrate specificities (12)(13)(14)(15). Glutamate formed by the transamination is regenerated to 2-oxoglutarate by alanine aminotransferasemediated transfer of the amino group to pyruvate (15), resulting in the formation of alanine as one of the end products (15, 16).…”

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Genetic Examination and Mass Balance Analysis of Pyruvate/Amino Acid Oxidation Pathways in the Hyperthermophilic Archaeon Thermococcus kodakarensis

et al. 2014

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bThe present study investigated the simultaneous oxidation of pyruvate and amino acids during H 2 -evolving growth of the hyperthermophilic archaeon Thermococcus kodakarensis. The comparison of mass balance between a cytosolic hydrogenase (HYH)-deficient strain (the ⌬hyhBGSL strain) and the parent strain indicated that NADPH generated via H 2 uptake by HYH was consumed by reductive amination of 2-oxoglutarate catalyzed by glutamate dehydrogenase. Further examinations were done to elucidate functions of three enzymes potentially involved in pyruvate oxidation: pyruvate formate-lyase (PFL), pyruvate:ferredoxin oxidoreductase (POR), and 2-oxoisovalerate:ferredoxin oxidoreductase (VOR) under the HYH-deficient background in T. kodakarensis. No significant change was observed by deletion of pflDA, suggesting that PFL had no critical role in pyruvate oxidation. The growth properties and mass balances of ⌬porDAB and ⌬vorDAB strains indicated that POR and VOR specifically functioned in oxidation of pyruvate and branched-chain amino acids, respectively, and the lack of POR or VOR was compensated for by promoting the oxidation of another substrate driven by the remaining oxidoreductase. The H 2 yields from the consumed pyruvate and amino acids were increased from 31% by the parent strain to 67% and 82% by the deletion of hyhBGSL and double deletion of hyhBGSL and vorDAB, respectively. Significant discrepancies in the mass balances were observed in excess formation of acetate and NH 3 , suggesting the presence of unknown metabolisms in T. kodakarensis grown in the rich medium containing pyruvate.T he euryarchaeal order Thermococcales, composed of two major genera, Thermococcus and Pyrococcus, comprises hyperthermophiles capable of growing at temperatures around 100°C (1, 2). Many members of the Thermococcales are anaerobic sulfur-reducing heterotrophs preferring proteinaceous substrates and utilize elemental sulfur (S 0 ) as a terminal electron acceptor evolving H 2 S. In the absence of S 0 , some of these hyperthermophiles can grow on carbohydrates and related compounds, evolving hydrogen gas (H 2 ) by using protons as a terminal electron acceptor (3).The energy-conserving metabolism of carbohydrates associated with H 2 evolution in the order Thermococcales has been well studied for Thermococcus kodakarensis and Pyrococcus furiosus (4). Pyruvate, added into the medium or generated from carbohydrates, is oxidized to acetyl coenzyme A (acetyl-CoA) by pyruvate: ferredoxin (Fd) oxidoreductase (POR) (5-8) and subsequently is converted to acetate concomitant with ATP synthesis by ADPforming acetyl-CoA synthetases (9, 10). In addition to pyruvate oxidation, peptides and amino acids also are dissimilated simultaneously (11). Amino acids, which are directly incorporated into the cells or formed by degradation of peptides, are deaminated to the corresponding 2-oxoacids using 2-oxoglutarate as an amino acceptor by a number of aminotransferases with different substrate specificities (12)(13)(14)(15). Glutamate formed by the transamina...

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“…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 distinct types of ferredoxin-and coenzyme A (CoA)-dependent 2-ketoacid oxidoreductases from these organisms have been characterized.…”

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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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Characterization of aromatic aminotransferases from the hyperthermophilic archaeon Thermococcus litoralis

Cited by 36 publications

References 42 publications

High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis

High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis

Genetic Examination and Mass Balance Analysis of Pyruvate/Amino Acid Oxidation Pathways in the Hyperthermophilic Archaeon Thermococcus kodakarensis

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

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