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Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius

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Abstract

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75°C and a 30-min half-inactivation temperature of ~90°C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of α-methyl and α-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

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References

  • Asada Y, Endo S, Inoue Y, Mamiya H, Hara A, Kunishima N, Matsunaga T (2009) Biochemical and structural characterization of a short-chain dehydrogenase/reductase of Thermus thermophilus HB8: a hyperthermostable aldose-1-dehydrogenase with broad substrate specificity. Chem Biol Interact 178:117–126

    Article  CAS  PubMed  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  CAS  PubMed  Google Scholar 

  • Chen L, Brügger K, Skovgaard M, Redder P, She Q, Torarinsson E, Greve B, Awayez M, Zibat A, Klenk HP, Garrett RA (2005) The genome of Sulfolobus acidocaldarius, a model organism of the Crenarchaeota. J Bacteriol 187:4992–4999

    Article  CAS  PubMed  Google Scholar 

  • Davies GE, Stark GR (1970) Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proc Natl Acad Sci USA 66:651–656

    Article  CAS  PubMed  Google Scholar 

  • Ema T, Ide S, Okita N, Sakai T (2008) Highly efficient chemoenzymatic synthesis of methyl (R)-o-chloromandelate, a key intermediate for clopidogrel, via asymmetric reduction with recombinant Escherichia coli. Adv Synth Catal 350:2039–2044

    Article  CAS  Google Scholar 

  • Filling C, Berndt KD, Benach J, Knapp S, Prozorovski T, Nordling E, Ladenstein R, Jörnvall H, Oppermann U (2002) Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J Biol Chem 277:25677–25684

    Article  CAS  PubMed  Google Scholar 

  • Fontana A, De Filippis V, Polverino de Laureto P, Scaramella E, Zambonin M (1998) Rigidity of thermophilic enzymes. In: Ballestreros A, Plou FJ, Iborra JL, Halling PJ (eds) Stability and stabilization in biocatalysis, vol 15. Elsevier, Amsterdam, The Netherlands, pp 277–294

    Google Scholar 

  • Gardinier KM, Leahy JW (1997) Enantiospecific total synthesis of the potent antitumor macrolides cryptophycins 1 and 8. J Org Chem 62:7098–7099

    Article  CAS  PubMed  Google Scholar 

  • Giordano A, Febbraio F, Russo C, Rossi M, Raia CA (2005) Evidence for co-operativity in coenzyme binding to tetrameric Sulfolobus solfataricus alcohol dehydrogenase and its structural basis: fluorescence, kinetic and structural studies of the wild-type enzyme and non-co-operative N249Y mutant. Biochem J 388:657–667

    Article  CAS  PubMed  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  CAS  PubMed  Google Scholar 

  • Höffken HW, Duong M, Friedrich T, Breuer M, Hauer B, Reinhardt R, Rabus R, Heider J (2006) Crystal structure and enzyme kinetics of the (S)-specific 1-phenylethanol dehydrogenase of the denitrifying bacterium strain EbN1. Biochemistry 45:82–93

    Article  PubMed  Google Scholar 

  • Hummel W (1999) Large-scale applications of NAD(P)-dependent oxidoreductases: recent developments. Trends Biotechnol 17:487–492

    Article  CAS  PubMed  Google Scholar 

  • Inoue K, Makino Y, Dairi T, Itoh N (2006) Gene cloning and expression of Leifsonia alcohol dehydrogenase (LSADH) involved in asymmetric hydrogen-transfer bioreduction to produce (R)-form chiral alcohols. Biosci Biotechnol Biochem 70:418–426

    Article  CAS  PubMed  Google Scholar 

  • John J, Crennell SJ, Hough DW, Danson MJ, Taylor GL (1994) The crystal structure of glucose dehydrogenase from Thermoplasma acidophilum. Structure 2:385–393

    Article  CAS  PubMed  Google Scholar 

  • Jones JB, Beck JF (1976) Applications of biochemical systems in organic chemistry. In: Jones JB, Sih CJ, Perlman D (eds) Techniques of chemistry series, part I, vol 10. Wiley, New York, NY, pp 248–401

    Google Scholar 

  • Jörnvall H (2008) Medium- and short-chain dehydrogenase/reductase gene and protein families: MDR and SDR gene and protein superfamilies. Cell Mol Life Sci 65:3873–3878

    Article  PubMed  Google Scholar 

  • Kallberg Y, Oppermann U, Jörnvall H, Persson B (2002) Short-chain dehydrogenases/reductases (SDRs). Eur J Biochem 269:4409–4417

    Article  CAS  PubMed  Google Scholar 

  • Kavanagh KL, Jörnvall H, Persson B, Oppermann U (2008) Medium- and short-chain dehydrogenase/reductase gene and protein families: the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes. Cell Mol Life Sci 65:3895–3906

    Article  CAS  PubMed  Google Scholar 

  • Keinan E, Hafely EK, Seth KK, Lamed R (1986) Thermostable enzymes in organic synthesis. Asymmetric reduction of ketones with alcohol dehydrogenase from Thermoanaerobium brockii. J Am Chem Soc 108:162–169

    Article  CAS  Google Scholar 

  • Korkhin Y, Kalb(Gilboa) AJ, Peretz M, Bogin O, Burstein Y, Frolow F (1998) NADP-dependent bacterial alcohol dehydrogenases: crystal structure, cofactor-binding and cofactor specificity of the ADHs of Clostridium beijerinckii and Thermoanaerobacter brockii. J Mol Biol 278:967–981

    Article  CAS  PubMed  Google Scholar 

  • Kroutil W, Mang H, Edegger K, Faber K (2004) Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols. Curr Opin Chem Biol 8:120–126

    Article  CAS  PubMed  Google Scholar 

  • Kwiecień RA, Ayadi F, Nemmaoui Y, Silvestre V, Zhang BL, Robins RJ (2009) Probing stereoselectivity and pro-chirality of hydride transfer during short-chain alcohol dehydrogenase activity: a combined quantitative 2H NMR and computational approach. Arch Biochem Biophys 482:42–51

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Leatherbarrow RJ (2004) GraFit Version 5.0.11. Erithacus Software Ltd., Horley, UK

    Google Scholar 

  • Liang ZX, Lee T, Resing KA, Ahn NG, Klinman JP (2004) Thermal-activated protein mobility and its correlation with catalysis in thermophilic alcohol dehydrogenase. Proc Natl Acad Sci USA 101:9556–9561

    Article  CAS  PubMed  Google Scholar 

  • Machielsen R, Uria AR, Kengen SW, van der Oost J (2006) Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldo–keto reductase superfamily. Appl Environ Microbiol 72:233–238

    Article  CAS  PubMed  Google Scholar 

  • Machielsen R, Leferink NG, Hendriks A, Brouns SJ, Hennemann HG, Daussmann T, van der Oost J (2008) Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to improve the production of (2S,5S)-hexanediol at moderate temperatures. Extremophiles 12:587–594

    Article  CAS  PubMed  Google Scholar 

  • Nie Y, Xu Y, Mu XQ, Wang HY, Yang M, Xiao R (2007) Purification, characterization, gene cloning, and expression of a novel alcohol dehydrogenase with anti-Prelog stereospecificity from Candida parapsilosis. Appl Environ Microbiol 73:3759–3764

    Article  CAS  PubMed  Google Scholar 

  • Niefind K, Müller J, Riebel B, Hummel W, Schomburg D (2003) The crystal structure of R-specific alcohol dehydrogenase from Lactobacillus brevis suggests the structural basis of its metal dependency. J Mol Biol 327:317–328

    Article  CAS  PubMed  Google Scholar 

  • Pennacchio A, Pucci B, Secundo F, La Cara F, Rossi M, Raia CA (2008) Purification and characterization of a novel recombinant highly enantioselective, short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus. Appl Environ Microbiol 74:3949–3958

    Article  CAS  PubMed  Google Scholar 

  • Pennacchio A, Giordano A, Esposito L, Langella E, Rossi M, Raia CA (2009) Insight into the Stereospecificity of Short-Chain Thermus thermophilus Alcohol Dehydrogenase Showing pro-S Hydride Transfer and Prelog Enantioselectivity. Protein Pept Lett (in press)

  • Persson B, Kallberg Y, Bray JE, Bruford E, Dellaporta SL, Favia AD, Duarte RG, Jörnvall H, Kavanagh KL, Kedishvili N, Kisiela M, Maser E, Mindnich R, Orchard S, Penning TM, Thornton JM, Adamski J, Oppermann U (2009) The SDR (short-chain dehydrogenase/reductase and related enzymes) nomenclature initiative. Chem Biol Interact 178:94–98

    Article  CAS  PubMed  Google Scholar 

  • Radianingtyas H, Wright PC (2003) Alcohol dehydrogenases from thermophilic and hyperthermophilic archaea and bacteria. FEMS Microbiol Rev 27:593–616

    Article  CAS  PubMed  Google Scholar 

  • Raia CA, Giordano A, Rossi M (2001) Alcohol dehydrogenase from Sulfolobus solfataricus. Methods Enzymol 331:176–195

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Schlieben NH, Niefind K, Muller J, Riebel B, Hummel W, Schomburg D (2005) Atomic resolution structures of R-specific alcohol dehydrogenase from Lactobacillus brevis provide the structural bases of its substrate and cosubstrate specificity. J Mol Biol 349:801–813

    Article  CAS  PubMed  Google Scholar 

  • Schneider-Bernlöhr H, Adolph H-W, Zeppezauer M (1986) Coenzyme stereospecificity of alcohol/polyol dehydrogenases: conservation of protein types vs. functional constraints. J Am Chem Soc 108:5573–5576

    Article  Google Scholar 

  • van der Oost J, Voorhorst WG, Kengen SW, Geerling AC, 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 

  • Zhao YH, Abraham MH, Zissimos AM (2003) Fast calculation of van der Waals volume as a sum of atomic and bond contributions and its application to drug compounds. J Org Chem 68:7368–7373

    Article  CAS  PubMed  Google Scholar 

  • Zhu D, Malik HT, Hua L (2006) Asymmetric ketone reduction by a hyperthermophilic alcohol dehydrogenase. The substrate specificity, enantioselectivity and tolerance of organic solvents. Tetrahedron: Asymmetry 17:3010–3014

    Article  CAS  Google Scholar 

  • Zhu D, Hyatt BA, Hua L (2009) Enzymatic hydrogen transfer reduction of α-chloro aromatic ketones catalyzed by a hyperthermophilic alcohol dehydrogenase. J Mol Catal B: Enzym 56:272–276

    Article  CAS  Google Scholar 

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Acknowledgments

This work was funded by FIRB (Fondo per gli Investimenti della Ricerca di Base) RBNE034XSW, and by the ASI project MoMa n. 1/014/06/0.

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Authors and Affiliations

  1. Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, 80131, Naples, Italy

    Angela Pennacchio, Mosè Rossi & Carlo A. Raia

  2. Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, 80078, Pozzuoli, Naples, Italy

    Assunta Giordano

  3. Centro Ricerche Oncologiche Mercogliano (CROM), Via A. Bianco, 83013, Mercogliano, Avellino, Italy

    Biagio Pucci

Authors
  1. Angela Pennacchio
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  2. Assunta Giordano
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  3. Biagio Pucci
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  4. Mosè Rossi
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  5. Carlo A. Raia
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Correspondence to Carlo A. Raia.

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Communicated by H. Santos.

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Pennacchio, A., Giordano, A., Pucci, B. et al. Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius . Extremophiles 14, 193–204 (2010). https://doi.org/10.1007/s00792-009-0298-3

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