Skip to main content
SpringerLink
Log in

Characterization of alcohol dehydrogenase from the haloalkaliphilic archaeon Natronomonas pharaonis

  • Note
  • Published:
Extremophiles Aims and scope Submit manuscript

Abstract

Alcohol dehydrogenase (ADH; EC: 1.1.1.1) is a key enzyme in production and utilization of ethanol. In this study, the gene encoding for ADH of the haloalkaliphilic archaeon Natronomonas pharaonis (NpADH), which has a 1,068-bp open reading frame that encodes a protein of 355 amino acids, was cloned into the pET28b vector and was expressed in Escherichia coli. Then, NpADH was purified by Ni-NTA affinity chromatography. The recombinant enzyme showed a molecular mass of 41.3 kDa by SDS-PAGE. The enzyme was haloalkaliphilic and thermophilic, being most active at 5 M NaCl or 4 M KCl and 70°C, respectively. The optimal pH was 9.0. Zn2+ significantly inhibited activity. The K m value for acetaldehyde was higher than that for ethanol. It was concluded that the physiological role of this enzyme is likely the catalysis of the oxidation of ethanol to acetaldehyde.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Bieger B, Essen LO, Oesterhelt D (2003) Crystal structure of halophilic dodecin. A novel, dodecameric flavin binding protein from Halobacterium salinarum. Structure 11:375–385

    Article  PubMed  CAS  Google Scholar 

  • Bonete MJ, Camacho ML, Cadenas E (1986) Purification and properties of NAD+-dependent glutamate dehydrogenase from Halobacterium halobium. Int J Biochem 18:785–789

    Article  CAS  Google Scholar 

  • Bonete MJ, Camacho ML, Cadenas E (1987) A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity. Int J Biochem 19:1149–1155

    Article  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Bräsen C, Schönheit P (2004) Regulation of acetate and acetyl-CoA converting enzymes during growth on acetate and/or glucose in the halophilic archaeon Haloarcula marismortui. FEMS Microbiol Lett 241:21–26

    Article  PubMed  CAS  Google Scholar 

  • Ceccarelli C, Liang ZX, Strickler M, Prehna G, Goldstein BM, Klinman JP, Bahnson BJ (2004) Crystal structure and amide H/D exchange of binary complexes of alcohol dehydrogenase from Bacillus stearothermophilus: Insight into thermostability and cofactor binding. Biochemistry 43:5266–5277

    Article  PubMed  CAS  Google Scholar 

  • Danson MJ, Hough DW (1997) The structural basis of protein halophilicity. Comp Biochem Physiol 117A:307–312

    Article  CAS  Google Scholar 

  • Demirjian DC, Moris-Varas F, Cassidy CS (2001) Enzymes from extremophiles. Curr Opin Chem Biol 5:144–151

    Article  PubMed  CAS  Google Scholar 

  • Dym O, Mevarech M, Sussman JL (1995) Structural features that stabilize halophilic malate dehydrogenase from an archaebacterium. Science 267:1344–1346

    Article  PubMed  CAS  Google Scholar 

  • Ebel C, Costenaro L, Pascu M, Faou P, Kernal B, Proust-De Martin F, Zaccai G (2002) Solvent interactions of halophilic malate dehydrogenase. Biochemistry 41:13234–13244

    Article  PubMed  CAS  Google Scholar 

  • Falb M, Pfeiffer F, Palm P, Rodewald K, Hickmann V, Tittor J, Oesterhelt D (2005) Living with two extremes: conclusions from the genome sequence of Natronomonas pharaonis. Genome Res 15:1336–1343

    Article  PubMed  CAS  Google Scholar 

  • Forrest GL, Gonzalez B (2000) Carbonyl reductases. Chem Biol Interact 129: 21–40

    Article  PubMed  CAS  Google Scholar 

  • Frolow F, Harel M, Sussman JL, Mevarech M, Shoham M (1996) Protein adaptation to a saturated salt environment: insights from the crystal structure of a halophilic 2Fe-2S ferredoxin. Nature Struct Biol 3:451–457

    Article  Google Scholar 

  • Fukuchi S, Yoshimune K, Wakayama M, Moriguchi M, Nishikawa K (2003) Unique amino acid composition of proteins in halophilic bacteria. J Mol Biol 327:347–357

    Article  PubMed  CAS  Google Scholar 

  • Irimia A, Ebel C, Madern D, Richard SB, Cosenza LW, Zaccai G, Vellieux FMD (2003) The oligomeric states of Haloarcula marismortui malate dehydrogenase are modulated by solvent components as shown by crystallographic and biochemical studies. J Mol Biol 326:859–873

    Article  PubMed  CAS  Google Scholar 

  • Jörnvall H, Persson B, Jeffery J (1987) Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases. Eur J Biochem 167:195–201

    Article  PubMed  Google Scholar 

  • Kamekura M (1998) Diversity of extremely halophilic bacteria. Extremophiles 2:289–295

    Article  PubMed  CAS  Google Scholar 

  • Konstantinidis K, Tebbe A, Klein C, Scheffer B, Aivaliotis M, Bisle B, Falb M, Pfeiffer F, Siedler F, Oesterhelt D (2007) Genome-wide proteomics of Natronomonas pharaonis. J Proteome Res 6:185–193

    Article  PubMed  CAS  Google Scholar 

  • Kumar S, Tamura K, Nei M (1994) MEGA: molecular evolutionary genetics analysis software for microcomputers. Comput Appl Biosci 10:189–191

    PubMed  CAS  Google Scholar 

  • Kushner DJ (1978) Life in high salt and solute concentrations. In: Kushner DJ (ed) Microbial life in extreme environments. Academic Press, London, pp 317–368

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Madern D, Ebel C, Zaccai G (2000) Halophilic adaptation of enzymes. Extremophiles 4:91–98

    Article  PubMed  CAS  Google Scholar 

  • Madern D, Camacho M, Rodriguez-Arnedo A, Bonete MJ, Zaccai G (2004) Salt-dependent studies of NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii. Extremophiles 8:377–384

    Article  PubMed  CAS  Google Scholar 

  • Margesin R, Schinner F (2001) Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles 5:73–83

    Article  PubMed  CAS  Google Scholar 

  • Ng WV, Kennedy SP, Mahairas GG, Berquist B, Pan M, Shukla HD, Lasky SR, Baliga NS, Thorsson V, Sbrogna J, Swartzell S, Weir D, Hall J, Dahl TA, Welti R, Goo YA, Leithauser B, Keller K, Cruz R, Danson MJ, Hough DW, Maddocks DG, Jablonski PE, Krebs MP, Angevine CM, Dale H, Isenbarger TA, Peck RF, Pohlschröder M, Spudich JL, Jung KW, Alam M, Freitas T, Hou S, Daniels CJ, Dennis PP, Omer AD, Ebhardt H, Lowe TM, Liang P, Riley M, Hood L, DasSarma S (2000) Genome sequence of Halobacterium species NRC-1. Proc Natl Acad Sci USA 97:12176–12181

    Article  PubMed  CAS  Google Scholar 

  • Oren A (1973) A thermophilic amyloglucosidase from Halobacterium sodomense, a halophilic bacterium from the Dead Sea. Curr Microbiol 8: 225–230

    Article  Google Scholar 

  • Padan E, Zilberstein D, Schuldiner S (1981) pH homeostasis in bacteria. Biochim Biophys Acta 650:151–166

    PubMed  CAS  Google Scholar 

  • Padan E, Bibi E, Ito M, Krulwich TA (2005) Alkaline pH homeostasis in bacteria: New insights. Biochim Biophys Acta 1717:67–88

    Article  PubMed  CAS  Google Scholar 

  • Pieper U, Kapadia G, Mevarech M, Herzberg O (1998) Structural features of halophilicity derived from the crystal structure of dihydrofolate reductase from the Dead Sea halophilic archaeon, Haloferax volcanii. Structure 6:75–88

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Richard SB, Madern D, Garcin E, Zaccai G (2000) Halophilic adaptation: novel solvent protein interactions observed in the 2.9 and 2.6 Å resolution structures of the wild type and a mutant of malate dehydrogenase from Haloarcula marismortui. Biochemistry 39:992–1000

    Article  PubMed  CAS  Google Scholar 

  • Saitou N, Nei M (1987) The neighbour joining method: a new tool for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  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 

  • Yamada Y, Fujiwara T, Sato T, Igarashi N, Tanaka N (2002) The 2.0 Å crystal structure of catalase-peroxidase from Haloarcula marismortui. Nat Struct Biol 9:691–695

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the grants from the Major State Basic Research Development Program of China (973 Program) (Grant No. 2004CB719604-3) and the National Natural Science Foundation of China (Grant No. 30670048).

Author information

Authors and Affiliations

  1. College of Life Sciences, Zhejiang University, Hangzhou, 310058, People’s Republic of China

    Yi Cao, Li Liao, Xue-wei Xu, Ce Wang, Xu-feng Zhu & Min Wu

  2. Institute of Life Sciences, and the Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel

    Aharon Oren

Authors
  1. Yi Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Li Liao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xue-wei Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Aharon Oren
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ce Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xu-feng Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Min Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Min Wu.

Additional information

Communicated by K. Horikoshi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cao, Y., Liao, L., Xu, Xw. et al. Characterization of alcohol dehydrogenase from the haloalkaliphilic archaeon Natronomonas pharaonis . Extremophiles 12, 471–476 (2008). https://doi.org/10.1007/s00792-007-0133-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00792-007-0133-7

Keywords

Search

Navigation