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Asymmetric reduction of diketones by two Gluconobacter oxydans oxidoreductases

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Abstract

Two genes encoding recombinant cytosolic oxidoreductases from Gluconobacter oxydans, gox0313 and gox0646, were heterologously expressed in Escherichia coli and the resulting proteins were purified and characterized. GOX0313 was identified as a medium-chain alcohol dehydrogenase, whereas GOX0646 was classified as a ketocarbonyl reductase. GOX0313 had a broad substrate spectrum and oxidized various primary alcohols. However, GOX0313 had a preference for substrate reduction, reducing many aldehydes and α-diketones. In contrast, GOX0646 had a narrow substrate spectrum and reduced α-diketones, preferring short-chain ketocarbonyls. Both enzymes regio- and stereospecifically reduced α-diketones to the corresponding (S)-hydroxy ketone, as shown by NMR. These products are difficult to produce chemically, requiring complicated protecting group chemistry. Furthermore, hydroxy ketones find industrial application in the production of pheromones, fragrances, flavors, and pharmaceuticals. Hence, these enzymes are interesting biocatalysts for the production of enantiomerically pure building blocks that are difficult to prepare chemically.

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References

  • Adachi O, Moonmangmee D, Toyama H, Yamada M, Shinagawa E, Matsushita K (2003) New developments in oxidative fermentation. Appl Microbiol Biotechnol 60:643–653

    CAS  Google Scholar 

  • Andersson A, Jordan D, Schneider G, Lindqvist Y (1996) Crystal structure of the ternary complex of 1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea with NADPH and an active-site inhibitor. Structure 4:1161–1170

    Article  CAS  Google Scholar 

  • Armarego WL (1979) Hydrogen transfer from 4-R and 4-S (4–3 H) NADH in the reduction of d, l-cis-6,7-dimethyl-6,7 (8 H) dihydropterin with dihydropteridine reductase from human liver and sheep liver. Biochem Biophys Res Commun 89:246–249

    Article  CAS  Google Scholar 

  • Arnold LJ, You KS, Allison WS, Kaplan NO (1976) Determination of hydride transfer stereospecificity of nicotinamide adenine-dinucleotide linked oxidoreductases by proton magnetic-resonance. Biochemistry 15:4844–4849

    Article  CAS  Google Scholar 

  • Ausubel FM (2002) Preparation and analysis of genomic DNA from bacteria. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Struhl K (eds) Current protocols in molecular biology, vol 5. Wiley, New York, pp 2–11

    Google Scholar 

  • Bel-Rhlid R, Fauve A, Veschambre H (1989) Synthesis of the pheromone components of the grape borer Xylotrechus pyrrhoderus by microbiological reduction of an α-diketone. J Org Chem 54:3221–3223

    Article  CAS  Google Scholar 

  • Benner SA, Nambiar KP, Chambers GK (1985) A stereochemical imperative in dehydrogenases—new data and criteria for evaluating function-based theories in bioorganic chemistry. J Am Chem Soc 107:5513–5517

    Article  CAS  Google Scholar 

  • Betz G, Warren JC (1968) Reaction mechanism and stereospecificity of 20 β-hydroxysteroid dehydrogenase. Arch Biochem Biophys 128:745–752

    Article  CAS  Google Scholar 

  • Blum H, Beier H, Gross HJ (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93–99

    Article  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  CAS  Google Scholar 

  • Cannio R, Rossi M, Bartolucci S (1994) A few amino acid substitutions are responsible for the higher thermostability of a novel NAD(+)-dependent bacillar alcohol dehydrogenase. Eur J Biochem 222(2):345–352

    Article  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  CAS  Google Scholar 

  • Chen J, Schenker S, Frosto TA, Henderson GI (1998) Inhibition of cytochrome c oxidase activity by 4-hydroxynonenal (HNE). Role of HNE adduct formation with the enzyme subunits. Biochim Biophys Acta 1380:336–344

    Article  CAS  Google Scholar 

  • Coppola GM, Schuster HF (1997) α-Hydroxy acids in enantioselective synthesis. Wiley-VCH, Weinheim

  • Crout HG, Morrey SM (1983) Synthesis of (R)- and (S)-acetoin (3-hydroxybutan-2-one). J Chem Soc, Perkin Trans 1:2435–2440

    Article  Google Scholar 

  • De Ley J, Gillis M, Swings J (1984) The genus Gluconobacter. In: Krieg NR, Holt JG (eds) Bergey’s manual of systematic bacteriology, vol 1. Williams and Wilkins, Baltimore, pp 267–278

    Google Scholar 

  • DeMaster EG, Shirota FN, Nagasawa HT (1986) Role of propiolaldehyde and other metabolites in the pargyline inhibition of rat-liver aldehyde dehydrogenase. Biochem Pharmacol 35:1481–1489

    Article  CAS  Google Scholar 

  • Deppenmeier U, Ehrenreich A (2009) Physiology of acetic acid bacteria in light of the genome sequence of Gluconobacter oxydans. J Mol Microbiol Biotechnol 16:69–80

    Article  CAS  Google Scholar 

  • Deppenmeier U, Hoffmeister M, Prust C (2002) Biochemistry and biotechnological applications of Gluconobacter strains. Appl Microbiol Biotechnol 60:233–242

    Article  CAS  Google Scholar 

  • Do Nascimento KH, Davies DD (1975) Stereospecificity of sequential nicotinamide-adenine dinucleotide-dependent oxidoreductases in relation to evolution of metabolic sequences. Biochem J 149:553–557

    CAS  Google Scholar 

  • Drauz K, Waldman H (eds) (2002) Enzyme catalysis in organic synthesis, vol 1-III. Wiley-VCH, Weinheim, Germany

    Google Scholar 

  • Fuganti C, Grasselli P (1982) Synthesis of the C14 chromanyl moiety of natural α-tocopherol (vitamin E). J Chem Soc Chem Commun 1982(4):205–206

    Article  Google Scholar 

  • Gala D, DiBedetto DJ, Clark JE, Murphy BL, Schuhmacher D, Steinman M (1996) Preparations of antifungal Sch 42427/SM 9164: Preparative chromatographic resolution, and total asymmetric synthesis via enzymatic preparation of chiral α-hydroxy arylketones. Tetrahedron Lett 37:611–614

    Article  CAS  Google Scholar 

  • Hall DR, Cork A, Phythian SJ, Chittamuru S, Jayarama BK, Venkatesha MG, Sreedharan K, Vinod Kumar PK, Seetharama HG, Naidu R (2006) Identification of components of male-produced pheromone of coffee white stemborer, Xylotrechus quadripes. J Chem Ecol 32:195–219

    Article  CAS  Google Scholar 

  • Hanke T, Richhardt J, Polen T, Sahm H, Bringer S, Bott M (2012) Influence of oxygen limitation, absence of the cytochrome bc1 complex and low pH on global gene expression in Gluconobacter oxydans 621 H using DNA microarray technology. J Biotechnol 157(3):359–372

    Article  CAS  Google Scholar 

  • Johansen L, Larsen SH, Størmer FC (1973) Diacetyl (acetoin) reductase from Aerobacter aerogenes—kinetic studies of reduction of diacetyl to acetoin. Eur J Biochem 34:97–99

    Article  CAS  Google Scholar 

  • Kavanagh KL, Jörnvall H, Persson B, Oppermann U (2008) The SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes. Cell Mol Life Sci 65:3895–3906

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) ClustalW and ClustalX version 2. Bioinformatics 23(21):2947–2948

    Article  CAS  Google Scholar 

  • Larsen SH, Størmer FC (1973) Diacetyl (acetoin) reductase from Aerobacter aerogenes—kinetic mechanism and regulation by acetate of reversible reduction of acetoin to 2,3-butanediol. Eur J Biochem 34:100–106

    Article  CAS  Google Scholar 

  • Lee JK, Koo BS, Kim SY, Hyun HH (2003) Purification and characterization of a novel mannitol dehydrogenase from a newly isolated strain of Candida magnoliae. Appl Environ Microbiol 69:4438–4447

    Article  CAS  Google Scholar 

  • Levin I, Meiri G, Peretz M, Burstein Y, Frolow F (2004) The ternary complex of Pseudomonas aeruginosa alcohol dehydrogenase with NADH and ethylene glycol. Protein Sci 13(6):1547–1556

    Article  CAS  Google Scholar 

  • Moore JC, Pollard DJ, Kosjek B, Devine PN (2007) Advances in the enzymatic reduction of ketones. Acc Chem Res 40:1412–1419

    Article  CAS  Google Scholar 

  • Mori K, Otsuka T (1985) Synthesis of (2S,3S)-2,3-octanediol and (S)-2-hydroxy-3-octanone, the male sex pheromone of the grape borer Xylotrechus pyrrhoderus. Tetrahedron 41:553–556

    Article  CAS  Google Scholar 

  • Mott JE, Grant RA, Ho YS, Platt T (1985) Maximizing gene expression from plasmid vectors containing the λPL promoter: strategies for overproducing transcription termination factor ρ. Proc Natl Acad Sci U S A 82:88–92

    Article  CAS  Google Scholar 

  • Neale AD, Scopes RK, Kelly JM, Wettenhall RE (1986) The two alcohol dehydrogenases of Zymomonas mobilis. Purification by differential dye ligand chromatography, molecular characterisation and physiological roles. Eur J Biochem 154(1):119–124

    Article  CAS  Google Scholar 

  • Palomo C, Oiarbide M, García JM (2012) α-Hydroxy ketones as useful templates in asymmetric reactions. Chem Soc Rev 41:4150–4164

    Article  CAS  Google Scholar 

  • Pauly S (1989) Permeability and diffusion data. In: Bandrup J, Immergut EH (eds) Polymer Handbook, 3rd edn. Wiley, New York, pp 435–449

    Google Scholar 

  • Prust C, Hoffmeister M, Liesegang H, Wiezer A, Fricke WF, Ehrenreich A, Gottschalk G, Deppenmeier U (2005) Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans. Nat Biotechnol 23:195–200

    Article  CAS  Google Scholar 

  • Rhainds M, Lan CC, King S, Gries R, Mo LZ, Gries G (2001) Pheromone communication and mating behaviour of coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae). Appl Entomol Zool 36:299–309

    Article  CAS  Google Scholar 

  • Saebel CE, Neureuther JM, Siemann S (2010) A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon. Anal Biochem 397:218–226

    Article  Google Scholar 

  • Sakoda H, Imanaka T (1992) Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH. J Bacteriol 174(4):1397–1402

    CAS  Google Scholar 

  • Salusjärvi T, Povelainen M, Hvorslev N, Eneyskaya EV, Kulminskaya AA, Shabalin KA, Neustroev KN, Kalkkinen N, Miasnikov AN (2004) Cloning of a gluconate/polyol dehydrogenase gene from Gluconobacter suboxydans IFO 12528, characterisation of the enzyme and its use for the production of 5-ketogluconate in a recombinant Escherichia coli strain. Appl Microbiol Biotechnol 65:306–314

    Article  Google Scholar 

  • Schedel M (2000) Regioselective oxidation of aminosorbitol with Gluconobacter oxydans, key reaction in the industrial 1-deoxynojirimycin synthesis. In: Kelly DR (ed) Biotechnology, vol 8b. Wiley-VCH, Weinheim, pp 295–308

    Chapter  Google Scholar 

  • Schweiger P, Gross H, Wesener S, Deppenmeier U (2008) Vinyl ketone reduction by three distinct Gluconobacter oxydans 621H enzymes. Appl Microbiol Biotechnol 80:955–1006

    Article  Google Scholar 

  • Schweiger P, Gross H, Deppenmeier U (2010) Characterization of two aldo-keto reductases from Gluconobacter oxydans 621H capable of regio- and stereoselective α-ketocarbonyl reduction. Appl Microbiol Biotechnol 87(4):1415–1426

    Article  CAS  Google Scholar 

  • Silber P, Chung H, Gargiulo P, Schulz H (1974) Purification and properties of a diacetyl reductase from Escherichia coli. J Bacteriol 118(3):919–927

    CAS  Google Scholar 

  • Steiner P, Sauer U (2001) Proteins induced during adaptation of Acetobacter aceti to high acetate concentrations. Appl Environ Microbiol 67:5474–5481

    Article  CAS  Google Scholar 

  • Uchida K, Stadtman ER (1993) Covalent attachment of 4-hydroxynonenal to glyceraldehyde-3-phosphate dehydrogenase. A possible involvement of intra- and intermolecular cross-linking reaction. J Biol Chem 268:6388–6393

    CAS  Google Scholar 

  • Vasic-Racki D (2000) Industrial biotransformation. In: Liese A, Seelbach K, Wandrey C (eds). Wiley-VCH, Weinheim Germany

  • Vidal I, Gonzalez J, Bernardo A, Martin R (1988) Purification and characterization of diacetyl-reducing enzymes from Staphylococcus aureus. Biochem J 251:461–466

    CAS  Google Scholar 

  • Willis LB, Walker GC (1998) Identification of the Rhizobium meliloti alcohol dehydrogenase gene (adhA) and heterologous expression in Alcaligenes eutrophus. Biochim Biophys Acta 1384(2):197–203

    Article  CAS  Google Scholar 

  • Yang IY, Hossain M, Miller H, Khullar S, Johnson F, Grollman A, Moriya M (2001) Responses to the major acrolein-derived deoxyguanosine adduct in Escherichia coli. J Biol Chem 276:9071–9076

    Article  CAS  Google Scholar 

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Acknowledgments

This project was supported by funds from Bundesministerium für Bildung und Forschung (BMBF, project no. 0315632A).

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Correspondence to Paul Schweiger.

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Schweiger, P., Gross, H., Zeiser, J. et al. Asymmetric reduction of diketones by two Gluconobacter oxydans oxidoreductases. Appl Microbiol Biotechnol 97, 3475–3484 (2013). https://doi.org/10.1007/s00253-012-4395-3

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