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J Bacteriol. Oct 1996; 178(20): 5890–5896.
PMCID: PMC178443

Characterization of a fourth type of 2-keto acid-oxidizing enzyme from a hyperthermophilic archaeon: 2-ketoglutarate ferredoxin oxidoreductase from Thermococcus litoralis.


Thermococcus litoralis is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 98 degrees C by fermenting peptides. It is known to contain three distinct ferredoxin-dependent, 2-keto acid oxidoreductases, which use pyruvate, aromatic 2-keto acids such as indolepyruvate, or branched-chain 2-keto acids such as 2-ketoisovalerate, as their primary substrates. We show here that T. litoralis also contains a fourth member of this family of enzymes, 2-ketoglutarate ferredoxin oxidoreductase (KGOR). In the presence of coenzyme A, KGOR catalyzes the oxidative decarboxylation of 2-ketoglutarate to succinyl coenzyme A and CO2 and reduces T. litoralis ferredoxin. The enzyme was oxygen sensitive (half-life of approximately 5 min) and was purified under anaerobic conditions. It had an M(r) of approximately 210,000 and appeared to be an octomeric enzyme (alpha2beta2gamma2delta2) with four different subunits with M(r)s of 43,000 (alpha), 29,000 (beta), 23,000 (gamma), and 10,000 (delta). The enzyme contained 0.9 mol of thiamine PPi and at least four [4Fe-4S] clusters per mol of holoenzyme as determined by metal analyses and electron paramagnetic resonance spectroscopy. Significant amounts of other metals (Cu, Zn, Mo, W, and Ni) were not present (<0.1 mol/mol of holoenzyme). Pure KGOR did not utilize other 2-keto acids, such as pyruvate, indolepyruvate, or 2-ketoisovalerate, as substrates, and the apparent Km values for 2-ketoglutarate, coenzyme A, T. litoralis ferredoxin, and thiamine PPi were approximately 250, 40, 8, and 9 microM, respectively. The enzyme was virtually inactive at 25 degrees C and exhibited optimal activity above 90 degrees C (at pH 8.0) and at pH 8.0 (at 80 degrees C). KGOR was quite thermostable, with a half-life at 80 degrees C (under anaerobic conditions) of about 2 days. An enzyme analogous to KGOR has been previously purified from a mesophilic archaeon, but the molecular properties of T. litoralis KGOR more closely resemble those of the other oxidoreductases from hyperthermophiles. In contrast to these enzymes, however, KGOR appears to have a biosynthetic function rather than a role in energy conservation.

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Selected References

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  • Aono S, Bryant FO, Adams MW. A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus. J Bacteriol. 1989 Jun;171(6):3433–3439. [PMC free article] [PubMed]
  • Beinert H. Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. Anal Biochem. 1983 Jun;131(2):373–378. [PubMed]
  • Beinert H, Thomson AJ. Three-iron clusters in iron-sulfur proteins. Arch Biochem Biophys. 1983 Apr 15;222(2):333–361. [PubMed]
  • Blamey JM, Adams MW. Purification and characterization of pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus. Biochim Biophys Acta. 1993 Jan 15;1161(1):19–27. [PubMed]
  • Blamey JM, Adams MW. Characterization of an ancestral type of pyruvate ferredoxin oxidoreductase from the hyperthermophilic bacterium, Thermotoga maritima. Biochemistry. 1994 Feb 1;33(4):1000–1007. [PubMed]
  • Brostedt E, Nordlund S. Purification and partial characterization of a pyruvate oxidoreductase from the photosynthetic bacterium Rhodospirillum rubrum grown under nitrogen-fixing conditions. Biochem J. 1991 Oct 1;279(Pt 1):155–158. [PMC free article] [PubMed]
  • Bryant FO, Adams MW. Characterization of hydrogenase from the hyperthermophilic archaebacterium, Pyrococcus furiosus. J Biol Chem. 1989 Mar 25;264(9):5070–5079. [PubMed]
  • Busse SC, La Mar GN, Yu LP, Howard JB, Smith ET, Zhou ZH, Adams MW. Proton NMR investigation of the oxidized three-iron clusters in the ferredoxins from the hyperthermophilic archae Pyrococcus furiosus and Thermococcus litoralis. Biochemistry. 1992 Dec 1;31(47):11952–11962. [PubMed]
  • Chen JS, Mortenson LE. Inhibition of methylene blue formation during determination of the acid-labile sulfide of iron-sulfur protein samples containing dithionite. Anal Biochem. 1977 May 1;79(1-2):157–165. [PubMed]
  • Conover RC, Kowal AT, Fu WG, Park JB, Aono S, Adams MW, Johnson MK. Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin. J Biol Chem. 1990 May 25;265(15):8533–8541. [PubMed]
  • Drake HL, Hu SI, Wood HG. Purification of five components from Clostridium thermoaceticum which catalyze synthesis of acetate from pyruvate and methyltetrahydrofolate. Properties of phosphotransacetylase. J Biol Chem. 1981 Nov 10;256(21):11137–11144. [PubMed]
  • Edelhoch H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 1967 Jul;6(7):1948–1954. [PubMed]
  • Gehring U, Arnon DI. Purification and properties of -ketoglutarate synthase from a photosynthetic bacterium. J Biol Chem. 1972 Nov 10;247(21):6963–6969. [PubMed]
  • Heider J, Mai X, Adams MW. Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea. J Bacteriol. 1996 Feb;178(3):780–787. [PMC free article] [PubMed]
  • Hoaki T, Nishijima M, Kato M, Adachi K, Mizobuchi S, Hanzawa N, Maruyama T. Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids. Appl Environ Microbiol. 1994 Aug;60(8):2898–2904. [PMC free article] [PubMed]
  • Hoaki T, Wirsen CO, Hanzawa S, Maruyama T, Jannasch HW. Amino Acid Requirements of Two Hyperthermophilic Archaeal Isolates from Deep-Sea Vents, Desulfurococcus Strain SY and Pyrococcus Strain GB-D. Appl Environ Microbiol. 1993 Feb;59(2):610–613. [PMC free article] [PubMed]
  • Hughes NJ, Chalk PA, Clayton CL, Kelly DJ. Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate:flavodoxin oxidoreductase from Helicobacter pylori. J Bacteriol. 1995 Jul;177(14):3953–3959. [PMC free article] [PubMed]
  • Kelly RM, Adams MW. Metabolism in hyperthermophilic microorganisms. Antonie Van Leeuwenhoek. 1994;66(1-3):247–270. [PubMed]
  • Kerscher L, Oesterhelt D. The catalytic mechanism of 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium. One-electron transfer at two distinct steps of the catalytic cycle. Eur J Biochem. 1981 Jun 1;116(3):595–600. [PubMed]
  • Kerscher L, Oesterhelt D. Purification and properties of two 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium. Eur J Biochem. 1981 Jun 1;116(3):587–594. [PubMed]
  • Kletzin A, Adams MW. Molecular and phylogenetic characterization of pyruvate and 2-ketoisovalerate ferredoxin oxidoreductases from Pyrococcus furiosus and pyruvate ferredoxin oxidoreductase from Thermotoga maritima. J Bacteriol. 1996 Jan;178(1):248–257. [PMC free article] [PubMed]
  • Kunow J, Linder D, Thauer RK. Pyruvate: ferredoxin oxidoreductase from the sulfate-reducing Archaeoglobus fulgidus: molecular composition, catalytic properties, and sequence alignments. Arch Microbiol. 1995 Jan;163(1):21–28. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Heider J, Ma K, Adams MW. Purification, characterization, and metabolic function of tungsten-containing aldehyde ferredoxin oxidoreductase from the hyperthermophilic and proteolytic archaeon Thermococcus strain ES-1. J Bacteriol. 1995 Aug;177(16):4757–4764. [PMC free article] [PubMed]
  • Ma K, Robb FT, Adams MW. Purification and characterization of NADP-specific alcohol dehydrogenase and glutamate dehydrogenase from the hyperthermophilic archaeon Thermococcus litoralis. Appl Environ Microbiol. 1994 Feb;60(2):562–568. [PMC free article] [PubMed]
  • Mai X, Adams MW. Indolepyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus. A new enzyme involved in peptide fermentation. J Biol Chem. 1994 Jun 17;269(24):16726–16732. [PubMed]
  • Mai X, Adams MW. Purification and characterization of two reversible and ADP-dependent acetyl coenzyme A synthetases from the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol. 1996 Oct;178(20):5897–5903. [PMC free article] [PubMed]
  • Meinecke B, Bertram J, Gottschalk G. Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum. Arch Microbiol. 1989;152(3):244–250. [PubMed]
  • Mukund S, Adams MW. Characterization of a novel tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon, Thermococcus litoralis. A role for tungsten in peptide catabolism. J Biol Chem. 1993 Jun 25;268(18):13592–13600. [PubMed]
  • Penttinen HK. Fluorometric determination of thiamine and its mono-, di-, and triphosphate esters. Methods Enzymol. 1979;62:58–59. [PubMed]
  • Pieulle L, Guigliarelli B, Asso M, Dole F, Bernadac A, Hatchikian EC. Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus. Biochim Biophys Acta. 1995 Jul 3;1250(1):49–59. [PubMed]
  • Riddles PW, Blakeley RL, Zerner B. Reassessment of Ellman's reagent. Methods Enzymol. 1983;91:49–60. [PubMed]
  • Robb FT, Park JB, Adams MW. Characterization of an extremely thermostable glutamate dehydrogenase: a key enzyme in the primary metabolism of the hyperthermophilic archaebacterium, Pyrococcus furiosus. Biochim Biophys Acta. 1992 Apr 17;1120(3):267–272. [PubMed]
  • Smith ET, Blamey JM, Adams MW. Pyruvate ferredoxin oxidoreductases of the hyperthermophilic archaeon, Pyrococcus furiosus, and the hyperthermophilic bacterium, Thermotoga maritima, have different catalytic mechanisms. Biochemistry. 1994 Feb 1;33(4):1008–1016. [PubMed]
  • Wahl RC, Orme-Johnson WH. Clostridial pyruvate oxidoreductase and the pyruvate-oxidizing enzyme specific to nitrogen fixation in Klebsiella pneumoniae are similar enzymes. J Biol Chem. 1987 Aug 5;262(22):10489–10496. [PubMed]
  • Weber K, Pringle JR, Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. [PubMed]
  • Wieland OH. The mammalian pyruvate dehydrogenase complex: structure and regulation. Rev Physiol Biochem Pharmacol. 1983;96:123–170. [PubMed]
  • Williams K, Lowe PN, Leadlay PF. Purification and characterization of pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis. Biochem J. 1987 Sep 1;246(2):529–536. [PMC free article] [PubMed]
  • Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4576–4579. [PMC free article] [PubMed]

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