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Resurrecting ancestral alcohol dehydrogenases from yeast


Modern yeast living in fleshy fruits rapidly convert sugars into bulk ethanol through pyruvate. Pyruvate loses carbon dioxide to produce acetaldehyde, which is reduced by alcohol dehydrogenase 1 (Adh1) to ethanol, which accumulates. Yeast later consumes the accumulated ethanol, exploiting Adh2, an Adh1 homolog differing by 24 (of 348) amino acids. As many microorganisms cannot grow in ethanol, accumulated ethanol may help yeast defend resources in the fruit1. We report here the resurrection of the last common ancestor2 of Adh1 and Adh2, called AdhA. The kinetic behavior of AdhA suggests that the ancestor was optimized to make (not consume) ethanol. This is consistent with the hypothesis that before the Adh1-Adh2 duplication, yeast did not accumulate ethanol for later consumption but rather used AdhA to recycle NADH generated in the glycolytic pathway. Silent nucleotide dating suggests that the Adh1-Adh2 duplication occurred near the time of duplication of several other proteins involved in the accumulation of ethanol, possibly in the Cretaceous age when fleshy fruits arose. These results help to connect the chemical behavior of these enzymes through systems analysis to a time of global ecosystem change, a small but useful step towards a planetary systems biology.

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Figure 1: The pathway by which yeast make, accumulate and then consume ethanol.
Figure 2: Maximum likelihood trees interrelating sequences determined in this work with sequences in the publicly available database.

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  1. Pretorius, I.S. Tailoring wine yeasts for the new millennium: novel approaches to the ancient art of winemaking. Yeast 16, 675–729 (2000).

    Article  CAS  Google Scholar 

  2. Thornton, J.W. Resurrecting ancient genes. Experimental analysis of extinct molecules. Nat. Rev. Genet. 5, 366–376 (2004).

    Article  CAS  Google Scholar 

  3. Boulton, B., Singleton, V.L., Bisson, L.F. & Kunkee, R.E. Yeast and biochemistry of ethanol fermentation. in Principles and Practices of Winemaking 139–172 (Chapman and Hall, New York, 1996).

    Chapter  Google Scholar 

  4. Fleet, G.H. & Heard, G.M. Yeast growth during fermentation. in Wine Microbiology and Biotechnology 27–54 (Harwood Academic Publishers, Chur, Switzerland, 1993).

    Google Scholar 

  5. McGovern, P.E. et al. Fermented beverages of pre- and proto-historic China. Proc. Natl. Acad. Sci. USA 101, 17593–17598 (2004).

    Article  CAS  Google Scholar 

  6. Wills, C. Production of yeast alcohol dehydrogenase isoenzymes by selection. Nature 261, 26 (1976).

    Article  CAS  Google Scholar 

  7. Fersht, A.R. Enzyme Structure and Mechanism. (W.H. Freeman, San Francisco, 1977).

    Google Scholar 

  8. Ellington, A.D. & Benner, S.A. Free energy differences between enzyme bound states. J. Theor. Biol. 127, 491–506 (1987).

    Article  CAS  Google Scholar 

  9. Stryer, L. Biochemistry 3rd edn. (Freeman, New York, 1988).

    Google Scholar 

  10. Swofford, D.L. PAUP* Phylogenetic Analysis Using Parsimony Version 4 (Sinauer Associates, Sunderland, Massachusetts, 1998).

    Google Scholar 

  11. Yang, Z.H. PAML. A program package for phylogenetic analysis by maximum likelihood. CABIOS 15, 555–556 (1997).

    Google Scholar 

  12. Thomson, J.M. Interpretive Proteomics: Experimental Paleogenetics as a Tool to Analyze Function and Discover Pathways in Yeast. Ph.D. dissertation. (University of Florida, Gainesville, 2002).

    Google Scholar 

  13. Weinhold, E.G., Glasfeld, A., Ellington, A.D. & Benner, S.A. Structural determinants of the stereospecificity of alcohol dehydrogenase from yeast. Proc. Natl. Acad. Sci. USA 88, 8420–8424 (1991).

    Article  CAS  Google Scholar 

  14. Ganzhorn, A.J., Green, D.W., Hershey, A.D., Gould, R.M. & Plapp, B.V. Kinetic characterization of yeast alcohol dehydrogenases. Amino acid residue 294 and substrate specificity. J. Biol. Chem. 262, 3754–3761 (1987).

    CAS  PubMed  Google Scholar 

  15. Segel, I.H. Enzyme Kinetics (Wiley, New York, 1975).

    Google Scholar 

  16. Kellis, M., Birren, B.W. & Lander, E.S. Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae . Nature 428, 617–624 (2004).

    Article  CAS  Google Scholar 

  17. Kreitman, M. & Akashi, H. Molecular evidence for natural selection. Ann. Rev. Ecol . Systematics 26, 403–422 (1995).

    Google Scholar 

  18. Benner, S.A., Caraco, M.D., Thomson, J.M. & Gaucher, E.A. Planetary biology. Paleontological, geological, and molecular histories of life. Science 293, 864–868 (2002).

    Article  Google Scholar 

  19. Sun, G. et al. Archaefructaceae, a new basal angiosperm family. Science 296, 899–904 (2002).

    Article  CAS  Google Scholar 

  20. Collinson, M.E. & Hooker, J.J. Fossil evidence of interactions between plants and plant-eating mammals. Philos. Trans. R. Soc. London Ser. B 333, 197–208 (1991).

    Article  CAS  Google Scholar 

  21. Fernandez-Espinar, M.T., Barrio, E. & Querol, A. Analysis of the genetic variability in the species of the Saccharomyces sensu stricto complex. Yeast 20, 1213–1226 (2003).

    Article  CAS  Google Scholar 

  22. Berbee, M.L. & Taylor, J.W. Dating the evolutionary radiations of the true fungi. Can. J. Bot. 71, 1114–1127 (1993).

    Article  Google Scholar 

  23. Benner, S.A. Interpretive proteomics. Finding biological meaning in genome and proteome databases. Adv. Enzyme Regul. 43, 271–359 (2003).

    Article  CAS  Google Scholar 

  24. Lynch, M. & Conery, J.S. The evolutionary fate and consequences of duplicate genes. Science 290, 1151–1155 (2000).

    Article  CAS  Google Scholar 

  25. Schaaff, I., Heinisch, J. & Zimmerman, F.K. Overproduction of glycolytic enzymes in yeast. Yeast 5, 285–290 (1989).

    Article  CAS  Google Scholar 

  26. Wolfe, K.H. & Shields, D.C. Molecular evidence for an ancient duplication of the entire yeast genome. Nature 387, 708–713 (2001).

    Article  Google Scholar 

  27. Ashburner, M. Speculations on the subject of alcohol dehydrogenase and its properties in Drosophila and other flies. Bioessays 20, 949–954 (1998).

    Article  CAS  Google Scholar 

  28. Barrett, P.M. & Willis, K.J. Did dinosaurs invent flowers? Dinosaur-angiosperm coevolution revisited. Biological Rev. 76, 411–447 (2001).

    Article  CAS  Google Scholar 

  29. Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F. & Cullin, C. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 21, 3329–3330 (1993).

    Article  CAS  Google Scholar 

  30. Bozzi, A., Saliola, M., Falcone, C., Bossa, F. & Martini, F. Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis . Biochim. Biophys. Acta 1339, 133–142 (1997).

    Article  CAS  Google Scholar 

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We thank A. Falcon and T. Barnash for their assistance. Funding was provided by the US National Research Council, the US National Aeronautics and Space Administration's Astrobiology Institute (E.A.G.) and the US National Aeronautics and Space Administration Exobiology program (S.A.B.).

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Correspondence to Steven A Benner.

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Supplementary information

Supplementary Fig. 1

Complementation of Adh 1/2 double minus with ancestral proteins. (PDF 4843 kb)

Supplementary Fig. 2

Trees showing f2 values for families with more than two paralogs. (PDF 87 kb)

Supplementary Table 1

Newly cloned and existing ADH genes used in the ancestral reconstruction. (PDF 163 kb)

Supplementary Note (PDF 130 kb)

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Thomson, J., Gaucher, E., Burgan, M. et al. Resurrecting ancestral alcohol dehydrogenases from yeast. Nat Genet 37, 630–635 (2005).

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