Use of the ND evidence code for Gene Ontology (GO) terms

SGD applied the ND (No biological Data available) evidence code to AAD3, formally recording that no experimental data supported a GO assignment in the affected aspect at the time of curation.

Electronic Gene Ontology annotations created by ARBA machine learning models

The single ARBA annotation on AAD3 (rule ARBA:ARBA00093038) assigns the specific molecular function GO:0047681 aryl-alcohol dehydrogenase (NADP+) activity, not a generic oxidoreductase term. Its condition set is nevertheless family-level rather than residue-level: one condition set combining a taxon condition with the FunFam structural signature 3.20.20.100:FF:000024 ("Aryl-alcohol dehydrogenase"), which AAD3 does match per its UniProt cross-references. Because the rule fires on fold/family membership and not on catalytic-site composition, it cannot see the AAD3-specific substitution of the catalytically essential AKR tyrosine (Cys73) and therefore propagates the ancestral family activity to a member shown biochemically to be inactive.

Disruption of seven hypothetical aryl alcohol dehydrogenase genes from Saccharomyces cerevisiae and construction of a multiple knock-out strain.

The seven S. cerevisiae AAD ORFs were identified in silico by high sequence similarity to the aryl-alcohol dehydrogenase of the lignin-degrading fungus Phanerochaete chrysosporium, and were named/annotated as putative aryl-alcohol dehydrogenases on that homology basis.

"By in silicio analysis, we have discovered that there are seven open reading frames (ORFs) in Saccharomyces cerevisiae whose protein products show a high degree of amino acid sequence similarity to the aryl alcohol dehydrogenase (AAD) of the lignin-degrading fungus Phanerochaete chrysosporium."
Deletion of the AAD genes, singly and in combination up to a septuple deletant, produced no detectable defect in aromatic-aldehyde reduction, arguing against a demonstrable, non-redundant aryl-alcohol dehydrogenase role for these genes (including AAD3) under the conditions tested.

"None of the knock-out strains revealed any mutant phenotype when tested for the degradation of aromatic aldehydes using both spectrophotometry and high performance liquid chromatography (HPLC)."

PMID:29079624

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

Of the seven purified recombinant yeast Aad proteins, only Aad4p and Aad14p reduced aryl-aldehydes with NADPH; purified Aad3p was inactive on the whole substrate panel.

"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)."
Aad3p carries a cysteine (Cys73) at the position of the catalytically essential tyrosine of the aldo/keto reductase catalytic tetrad, and reverting Cys73 to tyrosine did not restore any aryl-aldehyde reductase activity, indicating that AAD3 has accumulated additional inactivating changes and is pseudogenizing.

"However, alignment showed that in ScAad3p, a cysteine73 residue was present at the corresponding catalytic site tyrosine73. This substitution in ScAad3p could have been caused by a single nucleotide mutation from A218 to G218 of its coding sequence (Fig. 2 and S1). We therefore performed two site-directed mutagenesis experiments. In PcAad1p, the functional Tyr76 was mutated into Cys76 (TG227C to TA227C), whereas in ScAad3p, the Cys73 was replaced by Tyr73 (TA218C to TG218C). Following heterologous expression and purification, the activities of the recombinant proteins were assayed on a variety of aryl-aldehyde substrates. As expected, the PcAad1Tyr76Cysp variant was completely inactive on all of these substrates; however, correction of the missense mutation in ScAadCys73Tyrp failed to produce a functional enzyme (data not shown)."
Multicopy overexpression of AAD3 did not raise aryl-aldehyde reductase activity in crude extracts and did not improve growth on toxic aryl-aldehydes, in either wild-type or adh6 backgrounds.

"None of the transformants exhibited growth improvement on the four aryl-aldehydes, even those bearing AAD1 from P. chrysosporium. Indeed, while activity in BY4741 cell crude extracts expressing PcAad1p was 2-fold higher than that of the blank-plasmid control, activities in cells expressing ScAad3p, ScAad4p, and ScAd14p did not significantly differ (Fig. S7)."
The authors conclude that most of the BY4741 AAD genes, AAD3 included, are undergoing pseudogenization.

"Our observation that the majority of BY4741 AAD genes were undergoing pseudogenization prompted us to survey their distribution and sequence variation in diverse sequenced strains, including those adapted to the laboratory (BY4741 and Sigma1278b), to wine fermentation (EC1118, AWRI1631, AWRI796, Lalvin QA23, and VL3), to the brewing of beer (Fosters B and Fosters O) and sake (Kyokai no. 7), and to life as an opportunistic human pathogen (YJM789) (Table 3)."

PMID:27299603

Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts.

In an array-CGH survey of brewing strains, reduced copy number of several AAD genes (AAD3, AAD6, AAD10, AAD15 in one lager strain) correlated with redox imbalance and oxidative DNA damage; this is a strain-level correlation across the AAD family and provides no AAD3-specific mechanism.

"We found that decreased dosage of AAD genes was correlated with intracellular redox disequilibrium and oxidative DNA damage."