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Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium Thermotoga hypogea

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Abstract

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO2, H2, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe2+. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent Km values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.

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Abbreviations

ADH:

Alcohol dehydrogenase

CAPS:

3-(Cyclohexylamino)-1-propanesulfonic acid

EDTA:

Ethylenediaminetetraacetic acid

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

ITV-ICP-AES:

In-torch vaporization-inductively coupled plasma-atomic emission spectrometry

PIPES:

1,4-Piperazine-bis-(ethanesulfonic acid)

References

  • Antoine E, Rolland JL, Raffin JP, Dietrich J (1999) Cloning and over-expression in Escherichia coli of the gene encoding NADPH group III alcohol dehydrogenase from Thermococcus hydrothermomalis. Eur J Biochem 264:880–889

    Article  PubMed  CAS  Google Scholar 

  • Badiei HR, Smith AT, Karanassios V (2002) Rhenium-cup, in-torch vaporization (ITV) sample introduction for axially viewed ICP-AES and its application to the analysis of a microscopic, ng-weight solid sample. J Anal At Spectrom 17:1030–1036

    Article  CAS  Google Scholar 

  • Balk M, Weijma J, Stams AJM (2002) Thermotoga lettingae sp. nov., a novel thermophilic, methanol-degrading bacterium isolated from a thermophilic anaerobic reactor. Int J Syst Evol Microbiol 52:1361–1368

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Brinen LS, Canaves JM, Dai X, Deacon AM, Elsliger MA, Eshaghi S, Floyd R, Godzik A, Grittini C, Grzechnik SK, Guba C, Jaroszewski L, Karlak C, Klock HE, Koesema E, Kovarik JS, Kreusch A, Kuhn P, Lesley SA, McMullan D, McPhillips TM, Miller MA, Miller MD, Morse A, Moy K, Ouyang J, Robb A, Rodrigues K, Selby TL, Spraggon G, Stevens RC, van den Bedem H, Velasquez J, Vincent J, Wang X, West B, Wolf G, Taylor SS, Hodgson KO, Wooley J, Wilson IA (2002) Crystal structure of a zinc-containing glycerol dehydrogenase (TM0423) from Thermotoga maritima at 1.5 Å resolution. Proteins 50:371–374

    Article  CAS  Google Scholar 

  • Copeland A, Lucas S, Lapidus A, Barry K, Glavina del Rio T, Dalin E, Tice H, Bruce D, Pitluck S, Richardson P (2006a) Sequencing of the draft genome and assembly of Fervidobacterium nodosum Rt17-B1. US DOE Joint Genome Institute (JGI-PGF). http://www.genome.jgi-psf.org/draft_microbes/ferno/ferno.home.html accessed on 29 December, 2006

  • Copeland A, Lucas S, Lapidus A, Barry K, Glavina del Rio T, Dalin E, Tice H, Bruce D, Pitluck S, Richardson P (2006b) Sequencing of the draft genome and assembly of Thermotoga petrophila RKU-1. US DOE Joint Genome Institute (JGI-PGF). http://www.genome.jgi-psf.org/draft_microbes/thepr/thepr.home.html accessed on 29 December, 2006

  • Copeland A, Lucas S, Lapidus A, Barry K, Glavina del Rio T, Dalin E, Tice H, Bruce D, Pitluck S, Richardson P (2006c) Sequencing of the draft genome and assembly of Thermoanaerobacter ethanolicus X514. US DOE Joint Genome Institute (JGI-PGF). http://www.genome.jgi-psf.org/draft_microbes/theex/theex.home.html accessed on 29 December, 2006

  • Deutscher MP (1990) Guide to protein purification. Methods Enzymol 182:588–604

    Google Scholar 

  • Esposito L, Sica F, Raia CA, Giordano A, Rossi M, Mazzarella L, Zagari A (2002) Crystal structure of the alcohol dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus at 1.85 Å resolution. J Mol Biol 318:463–477

    Article  PubMed  CAS  Google Scholar 

  • Fardeau ML, Ollivier B, Patel BKC, Magot M, Thomas P, Rimbault A, Rocchiccioli F, Garcia JL (1997) Thermotoga hypogea sp. nov., a xylanolytic, thermophilic bacterium from an oil-producing well. Int J Syst Bacteriol 47:1013–1019

    Article  PubMed  CAS  Google Scholar 

  • Guagliardi A, Martino M, Iaccarino I, Rosa MD, Rossi M, Bartolucci S (1996) Purification and characterization of the alcohol dehydrogenase from a novel strain of Bacillus stearothermophilus growing at 70°C. Int J Biochem Cell Biol 28:239–246

    Article  PubMed  CAS  Google Scholar 

  • Guy JE, Isupov MN, Littlechild JA (2003) The structure of an alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix. J Mol Biol 331:1041–1051

    Article  PubMed  CAS  Google Scholar 

  • Hirakawa H, Kamiya N, Kawarabayashi Y, Nagamune T (2004) Properties of an alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix K1. J Biosci Bioeng 97:202–206

    PubMed  CAS  Google Scholar 

  • Ingram LO, Aldrich HC, Borges ACC, Causey TB, Martinez A, Morales F, Saleh A, Underwood SA, Yomano LP, York SW, Zaldivar J, Zhou S (1999) Enteric bacterial catalysts for fuel ethanol production. Biotechnol Prog 15:855–866

    Article  PubMed  CAS  Google Scholar 

  • Jochimsen B, Peinemann-Simon S, Völker H, Stüben D, Botz R, Stoffers P, Dando PR, Thomm M (1997) Stetteria hydrogenophila, gen. nov. and sp. nov., a novel mixotrophic sulfur-dependent crenarchaeote isolated from Milos, Greece. Extremophiles 1:67–73

    Article  PubMed  CAS  Google Scholar 

  • Kelly RM, Adams MWW (1994) Metabolism in hyperthermophilic microorganisms. Antonie Van Leeuwenhoek 66:247–270

    Article  PubMed  CAS  Google Scholar 

  • Kengen SWM, de Bok FAM, van Loo ND, Dijkema C, Stams AJM, de Vos WM (1994) Evidence for the operation of a novel Embden–Meyerhof pathway that involves ADP-dependent kinases during sugar fermentation by Pyrococcus furiosus. J Biol Chem 269:17537–17541

    PubMed  CAS  Google Scholar 

  • Kort R, Liebl W, Labedan B, Forterre P, Eggen RIL, de Vos WM (1997) Glutamate dehydrogenase from hyperthermophilic bacterium Thermotoga maritima: molecular characterization and phylogenetic implications. Extremophiles 1:52–60

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Littlechild JA, Guy JE, Isupov MN (2004) Hyperthermophilic dehydrogenase enzymes. Biochem Soc Trans 32:255–258

    Article  PubMed  CAS  Google Scholar 

  • Ma K, Adams MWW (1999) An unusual oxygen-sensitive, iron- and zinc-containing alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol 181:1163–1170

    PubMed  CAS  Google Scholar 

  • Ma K, Robb FT, Adams MWW (1994) Purification and characterization of NADP-specific alcohol dehydrogenase and glutamate dehydrogenase from the hyperthermophilic archaeon Thermococcus litoralis. Appl Environ Microbiol 60:562–568

    PubMed  CAS  Google Scholar 

  • Ma K, Loessner H, Heider J, Johnson MK, Adams MWW (1995) 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 177:4748–4756

    PubMed  CAS  Google Scholar 

  • Ma K, Hutchins A, Sung SJS, Adams MWW (1997) Pyruvate ferrodoxin oxidoreductase from the hyperthermophilic archaeon, Pyrococcus furiosus, functions as a CoA-dependent pyruvate decarboxylase. Proc Natl Acad Sci 94:9608–9613

    Article  PubMed  CAS  Google Scholar 

  • Nelson KE, Clayton R, Gill SR, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson J, Nelson WC, Ketchum KA, McDonald L, Utterback TR, Malek JA, Linher KD, Garrett MM, Stewart AM, Cotton MD, Pratt MS, Phillips CA, Richardson D, Heidelberg J, Sutton GG, Fleischmann RD, White O, Salzberg SL, Smith HO, Venter JC, Fraser CM (1999) Evidence for lateral gene transfer between archaea and bacteria from genome sequence of Thermotoga maritima. Nature 399:323–329

    Article  PubMed  CAS  Google Scholar 

  • van der Oost J, Voorhorst WGB, Kengen SWM, Geerling ACM, Wittenhorst V, Gueguen Y, de Vos WM (2001) Genetic and biochemical characterization of a short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus. Eur J Biochem 268:3062–3068

    Article  PubMed  Google Scholar 

  • Pan N, Imlay JA (2001) How does oxygen inhibit central metabolism in the obligate anaerobe Bacteroides thetaiotaomicron? Mol Microbiol 39:1562–1571

    Article  PubMed  CAS  Google Scholar 

  • Patel BKC, Morgan HW, Daniel RM (1985) Fervidobacterium nodosum gen. nov. and spec. nov. a new chemoorganotrophic, caldoactive, anaerobic bacterium. Arch Microbiol 141:63–69

    Article  CAS  Google Scholar 

  • Radianingtyas H, Wright PC (2003) Alcohol dehydrogenases from thermophilic and hyperthermophilic archaea and bacteria. FEMS Microbiol Rev 794:1–24

    Google Scholar 

  • Reid MF, Fewson CA (1994) Molecular characterization of microbial alcohol dehydrogenases. Crit Rev Microbiol 20:13–56

    PubMed  CAS  Google Scholar 

  • Rella R, Raia CA, Pensa M, Pisani FM, Gambacorta A, De Rosa M, Rossi M (1987) A novel archaebacterial NAD+-dependent alcohol dehydrogenase: purification and properties. Eur J Biochem 167:475–479

    Article  PubMed  CAS  Google Scholar 

  • Robb FT, Maeder DL (1998) Novel evolutionary histories and adaptive features of proteins from hyperthermophiles. Curr Opin Biotech 9:288–291

    Article  PubMed  CAS  Google Scholar 

  • Robb FT, Park JB, Adams MWW (1992) Characterization of an extremely thermostable glutamate dehydrogenase: a key enzyme in the primary metabolism of the hyperthermophilic archaebacterium, Pyrococcus furiosus. Biochim Biophys Acta 1120:267–272

    PubMed  CAS  Google Scholar 

  • Robb FT, Maeder DL, DiRuggiero J, Borges KM, Tolliday N (2001) Glutamate dehydrogenases from hyperthermophiles. In: Adams MWW, Kelly RM (eds) Methods in enzymology, vol 331. Academic, New York, pp 26–41

  • Sakuraba H, Goda S, Ohshima T (2004) Unique sugar metabolism and novel enzymes of hyperthermophilic archaea. Chem Rec 3:281–287

    Article  PubMed  CAS  Google Scholar 

  • Schönheit P, Schäfer T (1995) Metabolism of hyperthermophiles. World J Microbiol Biotechnol 11:26–57

    Article  Google Scholar 

  • Schwarzenbacher R, von Delft F, Canaves JM, Brinen LS, Dai X, Deacon AM, Elsliger MA, Eshaghi S, Floyd R, Godzik A, Grittini C, Grzechnik SK, Guba C, Jaroszewski L, Karlak C, Klock HE, Koesema E, Kovarik JE, Kreusch A, Kuhn P, Lesley SA, McMullan D, McPhillips TM, Miller MA, Miller MD, Morse A, Moy K, Ouyang J, Page R, Robb A, Rodrigues K, Selby TL, Spraggon G, Stevens RC, van den Bedem H, Velasquez J, Vincent J, Wang X, West B, Wolf G, Hodgson KO, Wooley J, Wilson IA (2004) Crystal structure of an iron-containing 1,3-propanediol dehydrogenase (TM0920) from Thermotoga maritima at 1.3 Å resolution. Proteins 54:174–177

    Article  PubMed  CAS  Google Scholar 

  • Scopes PK (1983) An iron-activated alcohol dehydrogenase. FEBS Lett 156:303–306

    Article  PubMed  CAS  Google Scholar 

  • Selig M, Xavier KB, Santos H, Schönheit P (1997) Comparative analysis of Embden–Meyerhof and Entner–Doudoroff glycolytic pathways in hyperthermophilic archaea and the bacterium Thermotoga. Arch Microbiol 167:217–232

    PubMed  CAS  Google Scholar 

  • Sheehan JJ (1994) Bioconversion for production of renewable transportation fuels in the United States: a strategic perspective. In: Himmel ME, Baker JO, Overend RP (eds) Enzymatic conversion of biomass for fuels production, ACS symposium series 566. American Chemical Society, Washington DC, pp 1–53

    Google Scholar 

  • Siebers B, Schönheit P (2005) Unusual pathways and enzymes of central carbohydrate metabolism in archaea. Curr Opin Microbiol 8:695–705

    Article  PubMed  CAS  Google Scholar 

  • Stetter KO (1989) Extremely thermophilic chemolithoautotrophic archaebacteria. In: Schlegel HG, Bowien B (eds) Autotrophic bacteria, Science Tech Publishers/Springer, Madison/Berlin, pp 167–176

  • Stetter KO (1996) Hyperthermophilic prokaryotes. FEMS Microbiol Rev 18:149–158

    Article  CAS  Google Scholar 

  • Ueda K, Yamashita A, Ishikawa J, Shimada M, Watsuji TO, Morimura K, Ikeda H, Hattori M, Beppu T (2004) Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalisms. Nucleic Acids Res 32:4937–4944

    Article  PubMed  CAS  Google Scholar 

  • de Vos WM, Kengen SWM, Voorhorst WGB, van der Oost J (1998) Sugar utilization and its control in hyperthermophile. Extremophiles 2:201–205

    Article  PubMed  Google Scholar 

  • Verhees CH, Kengen SW, Tuininga JE, Schut GJ, Adams MW, de Vos WM, van der Oost J (2003) The unique features of glycolytic pathways in archaea. Biochem J 375:231–246

    Article  PubMed  CAS  Google Scholar 

  • Wiegel J, Ljungdahl LG (1981) Thermoanaerobacter ethanolicus gen. nov., spec. nov., a new, extremely thermophilic, anaerobic bacterium. Arch Microbiol 128:343–348

    Article  CAS  Google Scholar 

  • Yang X, Ma K (2005) Purification and characterization of an NADH oxidase from extremely thermophilic anaerobic bacterium Thermotoga hypogea. Arch Microbiol 183:331–337

    Article  PubMed  CAS  Google Scholar 

  • Ziegenhorn J, Senn M, Bücher T (1976) Molar absorptivities of β-NADH and β-NADPH. Clin Chem 22(2):151–160

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by research grants from Ontario Ministry of Agriculture and Food, Rural Affairs, Natural Sciences and Engineering Research Council (Canada) and Canada Foundation for Innovation, and funds from the University of Waterloo to KM. We thank Feng Zhang for helping grow T. hypogea and Xianqin Yang for measuring glutamate dehydrogenase activities.

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  1. Ying Wang

    Present address: Xinjiang Agricultural University, Xinjiang, 830052, China

Authors and Affiliations

  1. Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada

    Xiangxian Ying, Ying Wang & Kesen Ma

  2. Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada

    Hamid R. Badiei & Vassili Karanassios

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  1. Xiangxian Ying
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  2. Ying Wang
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Correspondence to Kesen Ma.

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Ying, X., Wang, Y., Badiei, H.R. et al. Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium Thermotoga hypogea . Arch Microbiol 187, 499–510 (2007). https://doi.org/10.1007/s00203-007-0217-x

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