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. 1996 Feb;178(3):780–787. doi: 10.1128/jb.178.3.780-787.1996

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

J Heider 1, X Mai 1, M W Adams 1
PMCID: PMC177725  PMID: 8550513

Abstract

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.

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Selected References

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  1. Adams M. W. Biochemical diversity among sulfur-dependent, hyperthermophilic microorganisms. FEMS Microbiol Rev. 1994 Oct;15(2-3):261–277. doi: 10.1111/j.1574-6976.1994.tb00139.x. [DOI] [PubMed] [Google Scholar]
  2. Adams M. W. Enzymes and proteins from organisms that grow near and above 100 degrees C. Annu Rev Microbiol. 1993;47:627–658. doi: 10.1146/annurev.mi.47.100193.003211. [DOI] [PubMed] [Google Scholar]
  3. Andreotti G., Cubellis M. V., Nitti G., Sannia G., Mai X., Adams M. W., Marino G. An extremely thermostable aromatic aminotransferase from the hyperthermophilic archaeon Pyrococcus furiosus. Biochim Biophys Acta. 1995 Feb 22;1247(1):90–96. doi: 10.1016/0167-4838(94)00211-x. [DOI] [PubMed] [Google Scholar]
  4. Andreotti G., Cubellis M. V., Nitti G., Sannia G., Mai X., Marino G., Adams M. W. Characterization of aromatic aminotransferases from the hyperthermophilic archaeon Thermococcus litoralis. Eur J Biochem. 1994 Mar 1;220(2):543–549. doi: 10.1111/j.1432-1033.1994.tb18654.x. [DOI] [PubMed] [Google Scholar]
  5. Aono S., Bryant F. O., Adams M. W. A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus. J Bacteriol. 1989 Jun;171(6):3433–3439. doi: 10.1128/jb.171.6.3433-3439.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Beinert H., Thomson A. J. Three-iron clusters in iron-sulfur proteins. Arch Biochem Biophys. 1983 Apr 15;222(2):333–361. doi: 10.1016/0003-9861(83)90531-3. [DOI] [PubMed] [Google Scholar]
  7. Blamey J. M., Adams M. W. Characterization of an ancestral type of pyruvate ferredoxin oxidoreductase from the hyperthermophilic bacterium, Thermotoga maritima. Biochemistry. 1994 Feb 1;33(4):1000–1007. doi: 10.1021/bi00170a019. [DOI] [PubMed] [Google Scholar]
  8. Blamey J. M., Adams M. W. Purification and characterization of pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus. Biochim Biophys Acta. 1993 Jan 15;1161(1):19–27. doi: 10.1016/0167-4838(93)90190-3. [DOI] [PubMed] [Google Scholar]
  9. Blumentals I. I., Robinson A. S., Kelly R. M. Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus. Appl Environ Microbiol. 1990 Jul;56(7):1992–1998. doi: 10.1128/aem.56.7.1992-1998.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  11. 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. doi: 10.1042/bj2790155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bryant F. O., Adams M. W. Characterization of hydrogenase from the hyperthermophilic archaebacterium, Pyrococcus furiosus. J Biol Chem. 1989 Mar 25;264(9):5070–5079. [PubMed] [Google Scholar]
  13. Busse S. C., La Mar G. N., Yu L. P., Howard J. B., Smith E. T., Zhou Z. H., Adams M. W. 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. doi: 10.1021/bi00162a038. [DOI] [PubMed] [Google Scholar]
  14. Cannon M., Cannon F., Buchanan-Wollaston V., Ally D., Ally A., Beynon J. The nucleotide sequence of the nifJ gene of Klebsiella pneumoniae. Nucleic Acids Res. 1988 Dec 9;16(23):11379–11379. doi: 10.1093/nar/16.23.11379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chen J. S., Mortenson L. E. 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. doi: 10.1016/0003-2697(77)90390-6. [DOI] [PubMed] [Google Scholar]
  16. Consalvi V., Chiaraluce R., Politi L., Vaccaro R., De Rosa M., Scandurra R. Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus. Eur J Biochem. 1991 Dec 18;202(3):1189–1196. doi: 10.1111/j.1432-1033.1991.tb16489.x. [DOI] [PubMed] [Google Scholar]
  17. DiRuggiero J., Robb F. T., Jagus R., Klump H. H., Borges K. M., Kessel M., Mai X., Adams M. W. Characterization, cloning, and in vitro expression of the extremely thermostable glutamate dehydrogenase from the hyperthermophilic Archaeon, ES4. J Biol Chem. 1993 Aug 25;268(24):17767–17774. [PubMed] [Google Scholar]
  18. Dickinson J. R., Dawes I. W. The catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase in Saccharomyces cerevisiae. J Gen Microbiol. 1992 Oct;138(10):2029–2033. doi: 10.1099/00221287-138-10-2029. [DOI] [PubMed] [Google Scholar]
  19. Docampo R., Moreno S. N., Mason R. P. Free radical intermediates in the reaction of pyruvate:ferredoxin oxidoreductase in Tritrichomonas foetus hydrogenosomes. J Biol Chem. 1987 Sep 15;262(26):12417–12420. [PubMed] [Google Scholar]
  20. Heider J., Ma K., Adams M. W. 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. doi: 10.1128/jb.177.16.4757-4764.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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. doi: 10.1128/aem.60.8.2898-2904.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hoaki T., Wirsen C. O., Hanzawa S., Maruyama T., Jannasch H. W. 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. doi: 10.1128/aem.59.2.610-613.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hrdý I., Müller M. Primary structure and eubacterial relationships of the pyruvate:ferredoxin oxidoreductase of the amitochondriate eukaryote Trichomonas vaginalis. J Mol Evol. 1995 Sep;41(3):388–396. [PubMed] [Google Scholar]
  24. Kelly R. M., Adams M. W. Metabolism in hyperthermophilic microorganisms. Antonie Van Leeuwenhoek. 1994;66(1-3):247–270. doi: 10.1007/BF00871643. [DOI] [PubMed] [Google Scholar]
  25. Kengen S. W., de Bok F. A., van Loo N. D., Dijkema C., Stams A. J., de Vos W. M. Evidence for the operation of a novel Embden-Meyerhof pathway that involves ADP-dependent kinases during sugar fermentation by Pyrococcus furiosus. J Biol Chem. 1994 Jul 1;269(26):17537–17541. [PubMed] [Google Scholar]
  26. 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. doi: 10.1111/j.1432-1033.1981.tb05376.x. [DOI] [PubMed] [Google Scholar]
  27. 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. doi: 10.1111/j.1432-1033.1981.tb05377.x. [DOI] [PubMed] [Google Scholar]
  28. Kletzin A., Adams M. W. 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. doi: 10.1128/jb.178.1.248-257.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kunow J., Linder D., Thauer R. K. Pyruvate: ferredoxin oxidoreductase from the sulfate-reducing Archaeoglobus fulgidus: molecular composition, catalytic properties, and sequence alignments. Arch Microbiol. 1995 Jan;163(1):21–28. doi: 10.1007/BF00262199. [DOI] [PubMed] [Google Scholar]
  30. LOVENBERG W., BUCHANAN B. B., RABINOWITZ J. C. STUDIES ON THE CHEMICAL NATURE OF CLOSTRIDIAL FERREDOXIN. J Biol Chem. 1963 Dec;238:3899–3913. [PubMed] [Google Scholar]
  31. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  32. Lewin L. M., Wei R. Microassay of thiamine and its phosphate esters after separation by paper chromatography. Anal Biochem. 1966 Jul;16(1):29–35. doi: 10.1016/0003-2697(66)90077-7. [DOI] [PubMed] [Google Scholar]
  33. Ma K., Adams M. W. Sulfide dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus: a new multifunctional enzyme involved in the reduction of elemental sulfur. J Bacteriol. 1994 Nov;176(21):6509–6517. doi: 10.1128/jb.176.21.6509-6517.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ma K., Loessner H., Heider J., Johnson M. K., Adams M. W. Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase. J Bacteriol. 1995 Aug;177(16):4748–4756. doi: 10.1128/jb.177.16.4748-4756.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ma K., Robb F. T., Adams M. W. 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. doi: 10.1128/aem.60.2.562-568.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mai X., Adams M. W. 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] [Google Scholar]
  37. Meinecke B., Bertram J., Gottschalk G. Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum. Arch Microbiol. 1989;152(3):244–250. doi: 10.1007/BF00409658. [DOI] [PubMed] [Google Scholar]
  38. Mukund S., Adams M. W. 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] [Google Scholar]
  39. Mukund S., Adams M. W. Glyceraldehyde-3-phosphate ferredoxin oxidoreductase, a novel tungsten-containing enzyme with a potential glycolytic role in the hyperthermophilic archaeon Pyrococcus furiosus. J Biol Chem. 1995 Apr 14;270(15):8389–8392. doi: 10.1074/jbc.270.15.8389. [DOI] [PubMed] [Google Scholar]
  40. Mukund S., Adams M. W. The novel tungsten-iron-sulfur protein of the hyperthermophilic archaebacterium, Pyrococcus furiosus, is an aldehyde ferredoxin oxidoreductase. Evidence for its participation in a unique glycolytic pathway. J Biol Chem. 1991 Aug 5;266(22):14208–14216. [PubMed] [Google Scholar]
  41. Muller Y. A., Lindqvist Y., Furey W., Schulz G. E., Jordan F., Schneider G. A thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase, pyruvate oxidase and pyruvate decarboxylase. Structure. 1993 Oct 15;1(2):95–103. doi: 10.1016/0969-2126(93)90025-c. [DOI] [PubMed] [Google Scholar]
  42. Plaga W., Lottspeich F., Oesterhelt D. Improved purification, crystallization and primary structure of pyruvate:ferredoxin oxidoreductase from Halobacterium halobium. Eur J Biochem. 1992 Apr 1;205(1):391–397. doi: 10.1111/j.1432-1033.1992.tb16792.x. [DOI] [PubMed] [Google Scholar]
  43. Riddles P. W., Blakeley R. L., Zerner B. Reassessment of Ellman's reagent. Methods Enzymol. 1983;91:49–60. doi: 10.1016/s0076-6879(83)91010-8. [DOI] [PubMed] [Google Scholar]
  44. Robb F. T., Park J. B., Adams M. W. 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. doi: 10.1016/0167-4838(92)90247-b. [DOI] [PubMed] [Google Scholar]
  45. Shah V. K., Stacey G., Brill W. J. Electron transport to nitrogenase. Purification and characterization of pyruvate:flavodoxin oxidoreductase. The nifJ gene product. J Biol Chem. 1983 Oct 10;258(19):12064–12068. [PubMed] [Google Scholar]
  46. Smith E. T., Blamey J. M., Adams M. W. 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. doi: 10.1021/bi00170a020. [DOI] [PubMed] [Google Scholar]
  47. Thauer R. K., Jungermann K., Decker K. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev. 1977 Mar;41(1):100–180. doi: 10.1128/br.41.1.100-180.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Uyeda K., Rabinowitz J. C. Pyruvate-ferredoxin oxidoreductase. 3. Purification and properties of the enzyme. J Biol Chem. 1971 May 25;246(10):3111–3119. [PubMed] [Google Scholar]
  49. Wahl R. C., Orme-Johnson W. H. 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] [Google Scholar]
  50. Wang G. F., Kuriki T., Roy K. L., Kaneda T. The primary structure of branched-chain alpha-oxo acid dehydrogenase from Bacillus subtilis and its similarity to other alpha-oxo acid dehydrogenases. Eur J Biochem. 1993 May 1;213(3):1091–1099. doi: 10.1111/j.1432-1033.1993.tb17858.x. [DOI] [PubMed] [Google Scholar]
  51. Watrin L., Martin-Jezequel V., Prieur D. Minimal Amino Acid Requirements of the Hyperthermophilic Archaeon Pyrococcus abyssi, Isolated from Deep-Sea Hydrothermal Vents. Appl Environ Microbiol. 1995 Mar;61(3):1138–1140. doi: 10.1128/aem.61.3.1138-1140.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wieland O. H. The mammalian pyruvate dehydrogenase complex: structure and regulation. Rev Physiol Biochem Pharmacol. 1983;96:123–170. doi: 10.1007/BFb0031008. [DOI] [PubMed] [Google Scholar]
  53. Williams K., Lowe P. N., Leadlay P. F. Purification and characterization of pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis. Biochem J. 1987 Sep 1;246(2):529–536. doi: 10.1042/bj2460529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Woese C. R., Kandler O., Wheelis M. L. 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. doi: 10.1073/pnas.87.12.4576. [DOI] [PMC free article] [PubMed] [Google Scholar]

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