Characterization of NADH Oxidase/NADPH Polysulfide Oxidoreductase and Its Unexpected Participation in Oxygen Sensitivity in an Anaerobic Hyperthermophilic Archaeon

Kobori, Hiroki; Ogino, Masayuki; Orita, Izumi; Imanaka, Tadayuki; Fukui, Toshiaki

doi:10.1128/jb.00235-10

Cited by 21 publications

(28 citation statements)

References 40 publications

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“…However, its dimeric symmetry renders it ideal for inducing the transautophosphorylation events that underlie activation of eucaryal eIF2α [13]. It is also consistent with CoASH’s role as the major low molecular weight thiol component of the antioxidant defense systems in archaeal (and many bacterial) organisms [61,62]. …”

Section: Resultsmentioning

confidence: 77%

Activation of SsoPK4, an Archaeal eIF2α Kinase Homolog, by Oxidized CoA

et al. 2015

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The eukaryotic protein kinase (ePK) paradigm provides integral components for signal transduction cascades throughout nature. However, while so-called typical ePKs permeate the Eucarya and Bacteria, atypical ePKs dominate the kinomes of the Archaea. Intriguingly, the catalytic domains of the handful of deduced typical ePKs from the archaeon Sulfolobus solfataricus P2 exhibit significant resemblance to the protein kinases that phosphorylate translation initiation factor 2α (eIF2α) in response to cellular stresses. We cloned and expressed one of these archaeal eIF2α protein kinases, SsoPK4. SsoPK4 exhibited protein-serine/threonine kinase activity toward several proteins, including the S. solfataricus homolog of eIF2α, aIF2α. The activity of SsoPK4 was inhibited in vitro by 3ʹ,5ʹ-cyclic AMP (Ki of ~23 µM) and was activated by oxidized Coenzyme A, an indicator of oxidative stress in the Archaea. Activation enhanced the apparent affinity for protein substrates, Km, but had little effect on Vmax. Autophosphorylation activated SsoPK4 and rendered it insensitive to oxidized Coenzyme A.

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Section: Resultsmentioning

confidence: 77%

Activation of SsoPK4, an Archaeal eIF2α Kinase Homolog, by Oxidized CoA

et al. 2015

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“…Given these results, it seemed that expression of the SurR variants was incompatible with the survival of T. kodakarensis transformants using the established transformation procedure. One possibility was that constitutive repression of the S° regulon resulted in increased sensitivity to S° and/or oxygen (Kobori et al ., 2010) and so the transformation procedure was repeated using a S°‐free medium and strictly anaerobic conditions. This resulted in many pTS705, pTS709 and pTS710 transformants of T. kodakarensis TS1101, and sequencing plasmids isolated from representative transformants (Table 1) confirmed the presence of the intact TK1086 sequence with the cysteine to alanine mutation(s).…”

Section: Resultsmentioning

confidence: 99%

“…Glycolysis generates reduced ferredoxins (Fd red ) that must be re‐oxidized (Fd ox ) to maintain metabolic flow and conserve energy for growth. Based on analyses of culture products (Kanai et al ., 2005), the activities of enzymes and complexes purified from T. kodakarensis and closely related Thermococcales (Ma et al ., 2000; Sapra et al ., 2000; 2003; Ma and Adams, 2001a; Lee et al ., 2006; Chou et al ., 2007; Schut et al ., 2007; Lipscomb et al ., 2009; 2011; Clarkson et al ., 2010; Kobori et al ., 2010; Yang et al ., 2010), and the genome annotation (Fukui et al ., 2005), T. kodakarensis has several different systems to accomplish the regeneration of Fd ox from Fd red . Most of these predicted electron disposal pathways use sulfur (S°) as the terminal electron acceptor, generating H 2 S, but there are also pathways that result in alanine or H 2 production (Fig.…”

Section: Introductionmentioning

confidence: 99%

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Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production

Santangelo

Čuboňová

Reeve

2011

Molecular Microbiology

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Summary Hydrogen (H2) production by Thermococcus kodakarensis compares very favorably with the levels reported for the most productive algal, fungal and bacterial systems. T. kodakarensis can also consume H2 and is predicted to use several alternative pathways to recycle reduced cofactors, some of which may compete with H2 production for reductant disposal. To explore the reductant flux and possible competition for H2 production in vivo, T. kodakarensis TS517 was mutated to precisely delete each of the alternative pathways of reductant disposal, H2 production and consumption. The results obtained establish that H2 is generated predominantly by the membrane-bound hydrogenase complex (Mbh), confirm the essential role of the SurR (TK1086p) regulator in vivo, delineate the roles of sulfur (S°) regulon proteins and demonstrate that preventing H2 consumption results in a substantial net increase in H2 production. Constitutive expression of TK1086 (surR) from a replicative plasmid restored the ability of T. kodakarensis TS1101 (ΔTK1086) to grow in the absence of S° and stimulated H2 production, revealing a second mechanism to increase H2 production. Transformation of T. kodakarensis TS1101 with plasmids that express SurR variants constructed to direct the constitutive synthesis of the Mbh complex and prevent expression of the S°-regulon was only possible in the absence of S° and, under these conditions, the transformants exhibited wild-type growth and H2 production. With S° present, they grew slower but synthesized more H2 per unit biomass than T. kodakarensis TS517.

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“…PCR was carried out with KOD-Plus-ver.2 DNA polymerase (Toyobo, Osaka, Japan). Plasmids for gene disruption of hyhBGSL (pUD3-⌬hyh), pflDA (pUD3-⌬pfl), porDAB (pUD3-⌬por), and vorDAB (pUD3-⌬vor) in T. kodakarensis were constructed based on pUD3 harboring a pyrF marker cassette (41). pUD3-⌬hyh and pUD3-⌬vor were constructed as follows.…”

Section: Methodsmentioning

confidence: 99%

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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Characterization of NADH Oxidase/NADPH Polysulfide Oxidoreductase and Its Unexpected Participation in Oxygen Sensitivity in an Anaerobic Hyperthermophilic Archaeon

Cited by 21 publications

References 40 publications

Activation of SsoPK4, an Archaeal eIF2α Kinase Homolog, by Oxidized CoA

Activation of SsoPK4, an Archaeal eIF2α Kinase Homolog, by Oxidized CoA

Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production

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

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