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Resurrecting ancestral alcohol dehydrogenases from yeast.

ABSTRACT: 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 fruit. We report here the resurrection of the last common ancestor of Adh1 and Adh2, called Adh(A). The kinetic behavior of Adh(A) 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 Adh(A) 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.

SUBMITTER: Thomson JM

PROVIDER: S-EPMC3618678 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

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Resurrecting ancestral alcohol dehydrogenases from yeast.

Thomson J Michael JM Gaucher Eric A EA Burgan Michelle F MF De Kee Danny W DW Li Tang T Aris John P JP Benner Steven A SA

Nature genetics 20050501 6

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 fruit. We report here the resurrection of th ...[more]

PMID: 15864308

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Project description:Two datasets, the geologic record and the genetic content of extant organisms, provide complementary insights into the history of how key molecular components have shaped or driven global environmental and macroevolutionary trends. Changes in global physico-chemical modes over time are thought to be a consistent feature of this relationship between Earth and life, as life is thought to have been optimizing protein functions for the entirety of its approximately 3.8 billion years of history on the Earth. Organismal survival depends on how well critical genetic and metabolic components can adapt to their environments, reflecting an ability to optimize efficiently to changing conditions. The geologic record provides an array of biologically independent indicators of macroscale atmospheric and oceanic composition, but provides little in the way of the exact behaviour of the molecular components that influenced the compositions of these reservoirs. By reconstructing sequences of proteins that might have been present in ancient organisms, we can downselect to a subset of possible sequences that may have been optimized to these ancient environmental conditions. How can one use modern life to reconstruct ancestral behaviours? Configurations of ancient sequences can be inferred from the diversity of extant sequences, and then resurrected in the laboratory to ascertain their biochemical attributes. One way to augment sequence-based, single-gene methods to obtain a richer and more reliable picture of the deep past, is to resurrect inferred ancestral protein sequences in living organisms, where their phenotypes can be exposed in a complex molecular-systems context, and then to link consequences of those phenotypes to biosignatures that were preserved in the independent historical repository of the geological record. As a first step beyond single-molecule reconstruction to the study of functional molecular systems, we present here the ancestral sequence reconstruction of the beta-carbonic anhydrase protein. We assess how carbonic anhydrase proteins meet our selection criteria for reconstructing ancient biosignatures in the laboratory, which we term palaeophenotype reconstruction.This article is part of the themed issue 'Reconceptualizing the origins of life'.

Project description:1. Some aspects of the substrate specificities of liver and yeast alcohol dehydrogenases have been investigated with pentan-3-ol, heptan-4-ol, (+)-butan-2-ol, (+/-)-butan-2-ol, (+/-)-hexan-3-ol and (+/-)-octan-2-ol as potential substrates. The liver enzyme is active with all substrates tested, including both isomers of each optically active alcohol. In contrast, the yeast enzyme is completely inactive towards those secondary alcohols where both alkyl groups are larger than methyl and active with only the (+)-isomers of butan-2-ol and octan-2-ol. 2. The absence of stereospecificity of liver alcohol dehydrogenase towards optically active secondary alcohols and its broad specificity towards secondary alcohols in general are explained in terms of an alkyl-binding site that will react with a variety of alkyl groups and the ability of the enzyme to accommodate a fairly large unbound alkyl group in an active enzyme-NAD(+)-secondary alcohol ternary complex. The absolute optical specificity of the yeast enzyme towards n-alkylmethyl carbinols and its unreactivity towards pentan-3-ol, hexan-3-ol and heptan-4-ol are explained by its inability to accept alkyl groups larger than methyl in the unbound position in a viable ternary complex. 3. Comparison of the known configurations of the n-alkylmethyl carbinols and [1-(2)H]ethanol and [1-(3)H]geraniol, which have been used in stereospecificity studies with these enzymes by other workers, provides strong evidence for which alkyl group of the substrate is bound to the enzyme in the oxidation of n-alkylmethyl carbinols. The conclusions reached are, for butan-2-ol oxidation with the liver enzyme, confirmed by deductions from kinetic data obtained with (+)-butan-2-ol and a sample of butan-2-ol containing 66% of (-)-butan-2-ol. 4. Initial-rate parameters for the oxidations of (+)-butan-2-ol, 66% (-)-butan-2-ol and pentan-3-ol by NAD with liver alcohol dehydrogenase are presented. The data are completely consistent with a general mechanism of catalysis previously proposed for this enzyme.

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Resurrecting ancestral alcohol dehydrogenases from yeast.

Publications

Resurrecting ancestral alcohol dehydrogenases from yeast.

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