AMD2 (systematic name YDR242W) encodes a 549-residue probable amidase of Saccharomyces cerevisiae belonging to the amidase signature (AS) family (Pfam PF01425; InterPro IPR023631). Members of this family are amidohydrolases that use a conserved Ser-cisSer-Lys catalytic triad to hydrolyse carboxamide bonds, converting a carboxylic acid amide plus water into the corresponding carboxylate and ammonium. AMD2 retains the canonical catalytic triad and family sequence signature, and is assigned the generic amidase reaction (EC 3.5.1.4), but its physiological substrate, the specific reaction it catalyses in vivo, its subcellular localization, and the biological process it participates in have not been determined experimentally. Despite the family assignment, S. cerevisiae does not grow on acetamide as a nitrogen source and requires a heterologous acetamidase to do so, which argues against AMD2 acting as a classical acetamidase. It is a non-essential gene of currently uncharacterized biological role.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004040 amidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Domain-defensible generic molecular function. AMD2 is a bona fide member of the amidase signature (AS) family (Pfam PF01425; InterPro IPR023631; PROSITE PS00571) and retains the conserved Ser-cisSer-Lys catalytic triad (UniProt ACT_SITE 132, 209, 233). GO:0004040 corresponds exactly to the family/EC 3.5.1.4 reaction "a monocarboxylic acid amide + H2O = a monocarboxylate + NH4+", which is the generic parent activity. The annotation is appropriate AT THE GENERIC LEVEL; a specific-substrate amidase term would be over-annotation because the AS fold is substrate-promiscuous and no substrate has been determined for AMD2. Reason: Correct family- and fold-supported molecular function at the generic level. This is the single defensible functional statement for this dark gene and is retained as its core function. Supporting Evidence: UniProt:P22580 Pfam; PF01425; Amidase; 1. UniProt:P22580 InterPro; IPR023631; Amidase_dom. file:yeast/AMD2/AMD2-deep-research-falcon.md AMD2 belongs to the Amidase Signature (AS) superfamily |
| GO:0003674 molecular_function | ND GO_REF:0000015 | KEEP AS NON CORE | Summary: Root-level placeholder assigned by SGD with the ND (No biological Data) code, reflecting that no experimental molecular-function data existed at the time. It is superseded by the more specific IEA amidase-activity annotation above (which is itself only generic). Root MF terms carry no biological information and are not core. Reason: Uninformative root placeholder retained per GO conventions for ND annotations; not a curation target and not a core function. |
| GO:0005575 cellular_component | ND GO_REF:0000015 | KEEP AS NON CORE | Summary: Root-level placeholder assigned by SGD with the ND code because the subcellular localization of AMD2 is unknown. UniProt reports no SUBCELLULAR LOCATION and the sequence carries no predicted signal peptide, transit peptide, or transmembrane segment, so localization cannot be inferred with confidence. This remains an open CC knowledge gap (see knowledge_gaps). Reason: Uninformative root placeholder retained per GO conventions; localization is genuinely unknown, so no specific CC term can replace it. |
| GO:0008150 biological_process | ND GO_REF:0000015 | KEEP AS NON CORE | Summary: Root-level placeholder assigned by SGD with the ND code because the biological process AMD2 participates in is unknown. High-throughput deletion-phenotype data (decreased resistance to nutrient deprivation and to the antifungal miconazole) hint at a physiological consequence of loss but do not identify a specific process or pathway, and are not a basis for a specific BP term. This remains an open BP knowledge gap. Reason: Uninformative root placeholder retained per GO conventions; the in-vivo biological process is genuinely unknown. |
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Download this section (compressed HTML)Q: What monocarboxylic acid amide is the physiological substrate of AMD2, and is it a metabolic (nitrogen/carbon salvage) enzyme, a detoxification enzyme, or a signalling-lipid (fatty-acid amide) hydrolase?
Q: Where in the cell does AMD2 act? It has no predicted signal peptide, transit peptide, or transmembrane segment; is it cytosolic, or targeted to an organelle by a non-canonical signal?
Q: What is the mechanistic basis of the deletion phenotypes (decreased resistance to nutrient deprivation and to miconazole)? Do they reflect the enzyme's catalytic role or an indirect/moonlighting effect?
Experiment: Express and purify recombinant AMD2 and assay hydrolysis against a panel of monocarboxamide substrates (acetamide, propionamide, benzamide, medium/long-chain primary fatty-acid amides) measuring ammonium release and carboxylate product formation; determine kcat/Km for the best substrate(s). Include a catalytic-triad mutant (e.g. Ser209Ala) as a negative control.
Hypothesis: AMD2 is a catalytically active amidase whose product feeds nitrogen or carbon metabolism.
Type: enzyme activity assay
Experiment: Perform untargeted LC-MS metabolomics on wild-type versus amd2-delta strains under standard and nutrient-limiting conditions to identify metabolites that accumulate (candidate substrates) or are depleted (candidate products) upon deletion.
Hypothesis: Loss of AMD2 alters a specific amide-containing metabolite pool.
Type: comparative metabolomics
Experiment: Determine AMD2 subcellular localization by C-terminal fluorescent tagging at the endogenous locus and colocalization with organelle markers, and by subcellular fractionation.
Hypothesis: AMD2 localizes to a defined compartment consistent with its metabolic role.
Type: fluorescence localization
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The physiological substrate and the exact in-vivo reaction of AMD2 are undetermined. It is unknown which monocarboxylic acid amide is hydrolysed, so its generic amidase activity cannot be resolved to a specific molecular function. It is unlikely to be a classical acetamidase: wild-type S. cerevisiae S288C (which carries AMD2) cannot grow on acetamide as a nitrogen source and requires a heterologous acetamidase, though that growth assay reports on the whole genome rather than on AMD2 specifically.
OPEN BIOLOGY MF_DARK
What is known: AS-family membership (Pfam PF01425; InterPro IPR023631; PROSITE PS00571) and the conserved Ser-cisSer-Lys catalytic triad (UniProt ACT_SITE 132/209/233, annotated by similarity under ECO:0000250) support a generic carboxamide-hydrolase activity (EC 3.5.1.4; Rhea:12020) and an intact catalytic fold, without demonstrating catalytic competence. AMD2 sits in PANTHER subfamily SF11 ("amidase-related"), distinct from the fungal acetamidase subfamily SF9.
Significance: The AS fold is substrate-promiscuous across the family, so the generic IEA annotation is the ceiling of what sequence supports. The acetamide-growth data actively exclude the most obvious candidate substrate, leaving the true substrate open. Only experimental substrate identification can turn this into a defined molecular function.
What would resolve it: In-vitro substrate-panel enzymology on recombinant AMD2 (deliberately including non-acetamide amides such as fatty-acid primary amides and aryl amides) plus comparative metabolomics of wild-type versus amd2 deletion strains.
Provenance (the field's own admissions):
Gap: The biological process in which AMD2 participates is unknown. There is no pathway placement and no defined physiological role; the only functional data are non-specific high-throughput deletion phenotypes (decreased resistance to nutrient deprivation and to the antifungal miconazole).
OPEN BIOLOGY BP_DARK
What is known: AMD2 is a non-essential gene with a documented, reproducible generic amidase molecular function; a null mutant is viable, so any role is conditional or redundant.
Significance: Placing AMD2 in a metabolic or stress-response pathway would explain the deletion phenotypes and connect its enzymatic activity to yeast physiology.
What would resolve it: Epistasis/genetic-interaction analysis, condition-specific growth profiling tied to the identified substrate, and metabolomic pathway reconstruction.
Provenance (the field's own admissions):
Gap: The subcellular localization of AMD2 is unknown. It carries no experimentally determined location and the sequence has no predicted signal peptide, transit peptide, or transmembrane segment to guide a confident prediction.
OPEN BIOLOGY CC_DARK
What is known: UniProt reports no SUBCELLULAR LOCATION for AMD2; the entry annotates only the amidase domain and catalytic residues, with no targeting features.
Significance: Knowing where AMD2 acts would constrain candidate substrates and its metabolic context (e.g. cytosolic salvage versus organellar processing).
What would resolve it: Endogenous fluorescent tagging plus organelle-marker colocalization and subcellular fractionation.
Provenance (the field's own admissions):
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