Deciphering the Origin, Evolution, and Physiological Function of the Subtelomeric Aryl-Alcohol Dehydrogenase Gene Family in the Yeast Saccharomyces cerevisiae.

Jean-Marie Francois,Jin-Jing Zhang,Frank Rosenzweig,Emma R Master,Gustavo M De Billerbeck,Dong-Dong Yang

doi:10.1128/aem.01553-17

Abstract

ABSTRACTHomology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). Yeast AAD genes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes, AAD4 and AAD14, encode functional enzymes that reduce aliphatic and aryl-aldehydes concomitant with the oxidation of cofactor NADPH, and that Aad4p and Aad14p exhibit different substrate preference patterns. Other yeast AAD genes are undergoing pseudogenization. The 5′ sequence of AAD15 has been deleted from the genome. Repair of an AAD3 missense mutation at the catalytically essential Tyr73 residue did not result in a functional enzyme. However, ancestral-state reconstruction by fusing Aad6 with Aad16 and by N-terminal repair of Aad10 restores NADPH-dependent aryl-alcohol dehydrogenase activities. Phylogenetic analysis indicates that AAD genes are narrowly distributed in wood-saprophyte fungi and in yeast that occupy lignocellulosic niches. Because yeast AAD genes exhibit activity on veratraldehyde, cinnamaldehyde, and vanillin, they could serve to detoxify aryl-aldehydes released during lignin degradation. However, none of these compounds induce yeast AAD gene expression, and Aad activities do not relieve aryl-aldehyde growth inhibition. Our data suggest an ancestral role for AAD genes in lignin degradation that is degenerating as a result of yeast's domestication and use in brewing, baking, and other industrial applications.IMPORTANCE Functional characterization of hypothetical genes remains one of the chief tasks of the postgenomic era. Although the first Saccharomyces cerevisiae genome sequence was published over 20 years ago, 22% of its estimated 6,603 open reading frames (ORFs) remain unverified. One outstanding example of this category of genes is the enigmatic seven-member AAD family. Here, we demonstrate that proteins encoded by two members of this family exhibit aliphatic and aryl-aldehyde reductase activity, and further that such activity can be recovered from pseudogenized AAD genes via ancestral-state reconstruction. The phylogeny of yeast AAD genes suggests that these proteins may have played an important ancestral role in detoxifying aromatic aldehydes in ligninolytic fungi. However, in yeast adapted to niches rich in sugars, AAD genes become subject to mutational erosion. Our findings shed new light on the selective pressures and molecular mechanisms by which genes undergo pseudogenization.

Highlights

Homology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD)
We show that only two genes of this family (AAD4 and AAD14) encode enzymatic activities on aliphatic and aryl-aldehydes, whereas the other five putative members are being pseudogenized, a finding that informs our speculation about the evolutionary trajectory that created this subtelomeric gene family
Enzymatic activity of the seven purified yeast Aad recombinant proteins showed that only ScAad14p and ScAad4p were able to reduce a group of candidate aryl-aldehydes with the consumption of NADPH (Fig. 1), validating their predicted enzyme category as aryl-alcohol dehydrogenases (EC 1.1.1.90)

Summary

Introduction

Homology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). We showed that a homologue of yeast AAD, Phanerochaete chrysosporium Aad1p, can reduce aryl-aldehyde derivatives to their corresponding less-toxic alcohol forms [10] This result suggested that yeast Aad could serve to detoxify aromatic inhibitors produced in lignocellulosic ethanol production [22], as well as in the bioremediation of environmental pollutants, such as benzene, toluene, ethylbenzene and xylene (BTEX) derivatives [23]. To explore these possibilities and to enlarge our understanding of the function and phylogeny of the yeast Aad gene family, we performed detailed biochemical and molecular genetic analyses. We show that only two genes of this family (AAD4 and AAD14) encode enzymatic activities on aliphatic and aryl-aldehydes, whereas the other five putative members are being pseudogenized, a finding that informs our speculation about the evolutionary trajectory that created this subtelomeric gene family

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Conclusion