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.

Figure 1

The pathway by which yeast make, accumulate and then consume ethanol. Enzymes in red are associated with gene duplications that, according to the transition redundant exchange clock, arose nearly contemporaneously. The make-accumulate-consume pathway is boxed. The shunting of the carbon atoms from pyruvate into (and then out of, blue arrows) ethanol is energy-expensive, consuming a molecule of ATP (green) for every molecule of ethanol generated. This ATP is not consumed if pyruvate is oxidatively decarboxylated directly to acetyl-coenzyme A to enter the citric acid cycle directly (dashed arrow to the right). If dioxygen is available, the recycling of NADH does not need the acetaldehyde-to-ethanol reduction.

Figure 2

Maximum likelihood trees interrelating sequences determined in this work with sequences in the publicly available database. Shown are the two trees with the best (and nearly equal) ML scores using the following parameters estimated from the data: substitutions A↔C, A↔T, C↔G and G↔T = 1.00; A↔G = 2.92; and C↔T = 5.89; empirical base frequencies; and proportion of invariable sites and the shape parameter of the gamma distribution set to 0.33 and 1.31, respectively. The scale bar represents the number of substitutions per codon per unit evolutionary time. Single, double and triple asterisks represent bootstrap values greater than 50%, 70% and 90%, respectively.