ABSTRACTHomology searches indicate thatSaccharomyces cerevisiaestrain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). YeastAADgenes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes,AAD4andAAD14, 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 yeastAADgenes are undergoing pseudogenization. The 5′ sequence ofAAD15has been deleted from the genome. Repair of anAAD3missense mutation at the catalytically essential Tyr73residue 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 thatAADgenes are narrowly distributed in wood-saprophyte fungi and in yeast that occupy lignocellulosic niches. Because yeastAADgenes exhibit activity on veratraldehyde, cinnamaldehyde, and vanillin, they could serve to detoxify aryl-aldehydes released during lignin degradation. However, none of these compounds induce yeastAADgene expression, and Aad activities do not relieve aryl-aldehyde growth inhibition. Our data suggest an ancestral role forAADgenes in lignin degradation that is degenerating as a result of yeast's domestication and use in brewing, baking, and other industrial applications.IMPORTANCEFunctional characterization of hypothetical genes remains one of the chief tasks of the postgenomic era. Although the firstSaccharomyces cerevisiaegenome 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-memberAADfamily. 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 pseudogenizedAADgenes via ancestral-state reconstruction. The phylogeny of yeastAADgenes 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,AADgenes become subject to mutational erosion. Our findings shed new light on the selective pressures and molecular mechanisms by which genes undergo pseudogenization.
Ancestral State Reconstruction of the Apoptosis Machinery in the Common Ancestor of Eukaryotes
