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, indicating AMD2 is not 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 It is superseded by the more specific IEA amidase-activity annotation above (which is itself only generic).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.
|
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 specifically NOT a classical acetamidase: wild-type S. cerevisiae S288C (which carries AMD2) cannot grow on acetamide as a nitrogen source and requires a heterologous acetamidase.
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) firmly establish a generic carboxamide-hydrolase activity (EC 3.5.1.4; Rhea:12020) and a catalytically competent fold. 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):
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
AMD2 (systematic name YDR242W; synonyms AMDY, AMDY1) encodes a probable amidase in Saccharomyces cerevisiae (strain ATCC 204508 / S288c). The protein (UniProt accession P22580) is 598 amino acids in length and is classified under EC 3.5.1.4 (amidase / acylamide amidohydrolase). It is important to note that the functional annotation of AMD2 as an amidase is based on sequence homology and domain architecture rather than direct experimental biochemical characterization. The gene is located on chromosome IV of the S. cerevisiae genome.
The following table summarizes the key properties of AMD2:
| Property | Details |
|---|---|
| Gene Name | AMD2; synonyms reported in UniProt: AMDY, AMDY1 |
| Systematic Name | YDR242W |
| UniProt Accession | P22580 |
| Organism | Saccharomyces cerevisiae (strain ATCC 204508 / S288c), baker's yeast |
| EC Number | EC 3.5.1.4 (amidase / acylamide amidohydrolase), assigned as a probable function by annotation rather than direct biochemical validation for AMD2 (ramteke2013nitrile‐convertingenzymesan pages 10-13, ramteke2013nitrile‐convertingenzymesan pages 7-10) |
| Protein Family | Amidase signature (AS) family; broadly distributed amidohydrolases characterized by a conserved serine/glycine-rich amidase-signature region (moyacuevas2021beyondtheusual pages 1-2, ekici2008unconventionalserineproteases pages 10-11, ramteke2013nitrile‐convertingenzymesan pages 10-13) |
| Key Domains | Amidase_CS (IPR020556), Amidase_dom (IPR023631), AS_sf (IPR036928); consistent with UniProt/InterPro annotation and the AS-family structural framework (lee2015crystalstructureanalysis pages 3-4, moyacuevas2021beyondtheusual pages 1-2) |
| Predicted Catalytic Triad | Ser-cisSer-Lys; canonical catalytic configuration of amidase-signature enzymes (lee2015crystalstructureanalysis pages 3-4, moyacuevas2021beyondtheusual pages 1-2, ekici2008unconventionalserineproteases pages 10-11) |
| Predicted Function | Probable amidase predicted to hydrolyze amide bonds, yielding a carboxylic acid plus ammonia; however, the exact physiological substrate of AMD2 is unknown (ramteke2013nitrile‐convertingenzymesan pages 7-10, ramteke2013nitrile‐convertingenzymesan pages 10-13) |
| Subcellular Localization | Cytoplasm; consistent with database annotation and with a soluble non-membrane amidase, though direct AMD2-focused localization evidence is limited in the retrieved literature |
| Protein Length | 598 amino acids (UniProt) |
| Functional Status | Uncharacterized / probable; there is no direct experimental validation of AMD2 enzymatic activity or native substrate specificity in the retrieved AMD2-specific literature |
| Note on Acetamide | Wild-type S. cerevisiae S288C does not grow on acetamide as a sole nitrogen source, indicating AMD2 is unlikely to function as a classical acetamidase for acetamide under those conditions, despite amidase-family annotation (hamilton2020identificationofa pages 3-5, hamilton2020identificationofa pages 2-3) |
Table: This table condenses the verified identity, predicted biochemical role, family/domain architecture, and current evidence gaps for the yeast gene AMD2/YDR242W. It is useful for distinguishing annotation-based inference from experimentally demonstrated function, especially the key point that wild-type S. cerevisiae does not utilize acetamide.
AMD2 belongs to the Amidase Signature (AS) superfamily, a large and diverse group of amidohydrolases found across prokaryotes and eukaryotes, including archaea, bacteria, fungi, plants, insects, and mammals (moyacuevas2021beyondtheusual pages 1-2, ekici2008unconventionalserineproteases pages 10-11, ramteke2013nitrile‐convertingenzymesan pages 10-13). The defining structural feature of all AS family members is a conserved serine- and glycine-rich motif spanning approximately 50–130 amino acids, which contains an unconventional Ser-cisSer-Lys catalytic triad (moyacuevas2021beyondtheusual pages 1-2, ekici2008unconventionalserineproteases pages 10-11). This catalytic configuration distinguishes AS enzymes from classical serine proteases that employ a Ser-His-Asp triad.
AMD2 carries three InterPro-annotated domains consistent with AS family membership: the Amidase active site signature (Amidase_CS, IPR020556), the Amidase domain (Amidase_dom, IPR023631), and the Amidase superfamily fold (AS_sf, IPR036928), corresponding to Pfam family PF01425.
Based on the well-characterized catalytic mechanism of AS family enzymes, AMD2 is predicted to employ a Ser-cisSer-Lys catalytic triad. In this mechanism, the lysine residue functions as a general base (rather than histidine, as in classical serine proteases), and a bridging serine adopts an unusual cis-conformation that polarizes the catalytic serine nucleophile and forms a hydrogen-bonding network with the lysine (ekici2008unconventionalserineproteases pages 11-13). The catalytic cycle proceeds through: (i) nucleophilic attack by the catalytic serine on the carbonyl carbon of the amide bond, forming a tetrahedral intermediate; (ii) collapse to an acyl-enzyme intermediate with concomitant release of ammonia (or an amine); and (iii) hydrolysis of the acyl-enzyme intermediate to release the corresponding carboxylic acid and regenerate the free enzyme (moyacuevas2021beyondtheusual pages 2-4, ramteke2013nitrile‐convertingenzymesan pages 10-13). An oxyanion hole formed by glycine and serine backbone NH groups stabilizes the deprotonated carbonyl oxygen during catalysis (lee2015crystalstructureanalysis pages 3-4).
As an EC 3.5.1.4 enzyme, AMD2 is predicted to catalyze the general reaction:
R-CO-NH₂ + H₂O → R-COOH + NH₃
That is, the hydrolysis of a monocarboxylic acid amide to produce the corresponding carboxylic acid and ammonia (ramteke2013nitrile‐convertingenzymesan pages 7-10). Characterized EC 3.5.1.4 amidases typically act on short-chain aliphatic amides such as acetamide, propionamide, and butyramide (ramteke2013nitrile‐convertingenzymesan pages 10-13, fournand1998acyltransferactivity pages 2-3). However, as discussed below, the actual physiological substrate of AMD2 remains unknown.
A critical finding relevant to AMD2 function is that wild-type S. cerevisiae strain S288C cannot grow on acetamide as a sole nitrogen source (hamilton2020identificationofa pages 3-5, hamilton2020identificationofa pages 2-3). Hamilton et al. (2020) demonstrated that S. cerevisiae requires introduction of a heterologous acetamidase gene—either the Aspergillus nidulans amdS gene or the Yarrowia lipolytica YlAMD1 gene—to utilize acetamide (hamilton2020identificationofa pages 1-2, hamilton2020identificationofa pages 3-5). Control transformations lacking these heterologous genes showed no growth on acetamide plates (hamilton2020identificationofa pages 2-3). This directly demonstrates that AMD2, despite its sequence-based annotation as a probable amidase, does not confer functional acetamidase activity sufficient for growth on acetamide under standard conditions.
This observation is consistent with the known diversity of AS family substrates. The AS superfamily encompasses enzymes with vastly different substrate specificities, as illustrated in the following table:
| AS Family Member | Organism | Substrate | Biological Role |
|---|---|---|---|
| AMD2/YDR242W | Saccharomyces cerevisiae | Unknown (probable amide substrate[s]) | Probable amidase; exact physiological function remains uncharacterized, and wild-type S. cerevisiae does not grow on acetamide as sole nitrogen source, so its substrate cannot be assumed to be acetamide (hamilton2020identificationofa pages 3-5, hamilton2020identificationofa pages 2-3, moyacuevas2021beyondtheusual pages 1-2, ekici2008unconventionalserineproteases pages 10-11) |
| FAAH | Mammals | N-acylethanolamines (e.g., anandamide) | Endocannabinoid / fatty acid amide signaling termination by hydrolysis of NAEs (moyacuevas2021beyondtheusual pages 1-2, ekici2008unconventionalserineproteases pages 10-11, moyacuevas2021beyondtheusual pages 2-4) |
| AMI1 | Arabidopsis thaliana | Indole-3-acetamide | Auxin (IAA) biosynthesis via IAM hydrolysis (moyacuevas2021beyondtheusual pages 1-2, moyacuevas2021beyondtheusual pages 2-4) |
| amdS | Aspergillus nidulans | Acetamide | Nitrogen/carbon utilization from acetamide; classic fungal acetamidase used as a selectable marker (hamilton2020identificationofa pages 1-2, ramteke2013nitrile‐convertingenzymesan pages 10-13) |
| MAE2 (Malonamidase E2) | Bradyrhizobium japonicum | Malonamate | Nitrogen metabolism in symbiosis; converts malonamate to malonate and ammonia (lee2015crystalstructureanalysis pages 3-4, moyacuevas2021beyondtheusual pages 2-4) |
| Peptide amidase (PAM) | Stenotrophomonas maltophilia | C-terminal peptide amides | Peptide processing via selective hydrolysis of C-terminal amide bonds (lee2015crystalstructureanalysis pages 3-4, moyacuevas2021beyondtheusual pages 2-4) |
| Aryl acylamidase (AAA) | Bacteria | Aryl acylamides | Likely detoxification / specialized amide hydrolysis; substrate pocket studies show AS-family structural adaptation for aromatic amides (lee2015crystalstructureanalysis pages 3-4) |
| YlAMD1 | Yarrowia lipolytica | Acetamide | Nitrogen utilization; experimentally validated major acetamidase enabling growth on acetamide (hamilton2020identificationofa pages 3-5, hamilton2020identificationofa pages 1-2, hamilton2020identificationofa pages 2-3) |
| Note | AS family overview | Diverse amide-containing substrates | This comparison highlights the broad substrate range of amidase-signature enzymes; therefore, AMD2 sequence membership in the AS family does not by itself justify assigning acetamide as its native substrate (lee2015crystalstructureanalysis pages 3-4, moyacuevas2021beyondtheusual pages 1-2, ekici2008unconventionalserineproteases pages 10-11, ramteke2013nitrile‐convertingenzymesan pages 10-13) |
Table: This table compares representative amidase signature family enzymes across taxa to show how widely their substrates and biological roles vary. It is useful for interpreting AMD2 cautiously: family membership supports amidase-like chemistry, but not a specific substrate such as acetamide.
Thus, AMD2 may hydrolyze a different amide substrate entirely, may require specific induction conditions, or may have very low activity toward acetamide. Its physiological substrate in S. cerevisiae remains to be determined experimentally.
AMD2/YDR242W is annotated as localizing to the cytoplasm based on the global GFP localization study of S. cerevisiae proteins conducted by Huh et al. (2003, Nature 425:686–691). The protein has no predicted signal peptide or transmembrane domains, which is consistent with a soluble cytoplasmic enzyme. This localization pattern is typical of many AS family members, including plant AMI1 and bacterial malonamidase E2, which are soluble intracellular enzymes (moyacuevas2021beyondtheusual pages 2-4). In contrast, the mammalian FAAH is an integral membrane protein, representing an atypical case within the family (moyacuevas2021beyondtheusual pages 1-2).
The most intuitive pathway for an amidase in S. cerevisiae would be nitrogen metabolism, where hydrolysis of an amide substrate releases ammonia that can be assimilated by glutamate dehydrogenase or glutamine synthetase (schwardmann2024prospectsofformamide pages 5-6, ramteke2013nitrile‐convertingenzymesan pages 7-10). In fungi such as A. nidulans, the acetamidase amdS enables utilization of acetamide as a nitrogen source and is subject to nitrogen metabolite repression (hamilton2020identificationofa pages 1-2, ramteke2013nitrile‐convertingenzymesan pages 10-13). However, as noted above, S. cerevisiae lacks functional acetamidase activity and cannot use acetamide as a nitrogen source (hamilton2020identificationofa pages 3-5, hamilton2020identificationofa pages 2-3), so AMD2 likely does not participate in acetamide catabolism.
S. cerevisiae has been shown to contain N-acylethanolamines (NAEs) and their phospholipid precursors (N-acylphosphatidylethanolamines), and a yeast homolog of NAPE-PLD contributes to NAE biosynthesis—deletion of this homolog reduced saturated and monounsaturated NAE levels by approximately 60% (leung2006inactivationofnacyl pages 5-7). Given that FAAH, a well-characterized AS family member, is the principal enzyme responsible for NAE degradation in mammals (moyacuevas2021beyondtheusual pages 1-2, ekici2008unconventionalserineproteases pages 10-11), it is plausible that AMD2 could function in NAE metabolism in yeast. However, this hypothesis has not been tested experimentally for AMD2.
AS family members across organisms catalyze a remarkably diverse array of reactions, including hydrolysis of malonamate to malonate and ammonia (malonamidase E2), C-terminal amide processing of peptides (peptide amidase), and hydrolysis of 6-aminohexanoate cyclic dimers (NylA) (lee2015crystalstructureanalysis pages 3-4, moyacuevas2021beyondtheusual pages 2-4). The true physiological substrate and pathway context of AMD2 may correspond to an as-yet-unidentified amide-containing metabolite in S. cerevisiae.
Crystal structures of AS family members reveal a conserved overall fold in which the amidase signature region contains the catalytic triad and an oxyanion hole formed by conserved TGGS motifs (lee2015crystalstructureanalysis pages 3-4). Substrate specificity is determined by structural variations in loop regions flanking the active site: particularly loop1 (between β2 and α4) and loop2 (between β7 and β8), which together with an α-helix form the substrate-binding pocket (lee2015crystalstructureanalysis pages 3-4). The spatial orientation and amino acid composition of these loops vary significantly among AS family members, accounting for their diverse substrate preferences.
The AS family is one of the largest families of amidohydrolases, rivaling the classical serine protease families in number of members (ekici2008unconventionalserineproteases pages 10-11). The family is found from archaea (e.g., Pyrococcus, Sulfolobus) through bacteria, fungi, plants, and mammals, indicating ancient evolutionary origins (ekici2008unconventionalserineproteases pages 10-11, ramteke2013nitrile‐convertingenzymesan pages 10-13). AMD2 in S. cerevisiae has a paralog, AMD1 (YCR025C), though neither gene product has been biochemically characterized. The presence of amidase family genes in S. cerevisiae despite the organism's inability to utilize common amides like acetamide suggests that these enzymes may serve specialized metabolic functions unrelated to simple aliphatic amide catabolism.
The AMD2 gene is non-essential; deletion of YDR242W does not cause lethality or obvious growth defects under standard laboratory conditions (based on the S. cerevisiae genome-wide deletion collection). The amd2Δ mutant has not been specifically reported to show significant phenotypes in large-scale deletion screens, though this may reflect the lack of targeted assay conditions that would reveal its function.
AMD2 (YDR242W) is a poorly characterized member of the amidase signature (AS) enzyme superfamily in S. cerevisiae. Its annotation as a "probable amidase" (EC 3.5.1.4) is based on sequence homology to characterized AS family members, which share a conserved Ser-cisSer-Lys catalytic triad and catalyze the hydrolysis of diverse amide bonds. The protein is predicted to localize to the cytoplasm as a soluble enzyme. Critically, wild-type S. cerevisiae cannot grow on acetamide as a nitrogen source (hamilton2020identificationofa pages 3-5, hamilton2020identificationofa pages 2-3), which demonstrates that AMD2 does not function as a classical acetamidase despite its family assignment. The true physiological substrate and biological pathway of AMD2 remain experimentally undetermined. Potential roles—inferred from other AS family members—include hydrolysis of lipid amides (analogous to FAAH), specialized amide metabolites, or other uncharacterized amide-containing compounds in yeast metabolism. Biochemical characterization of recombinant AMD2, including substrate profiling, would be necessary to resolve its function. This gene represents a clear example of the challenges in functional annotation of genes assigned "probable" functions based solely on domain homology, particularly within enzyme superfamilies that exhibit broad substrate diversity.
References
(ramteke2013nitrile‐convertingenzymesan pages 10-13): Pramod W. Ramteke, Navodita G. Maurice, Babu Joseph, and Bharat J. Wadher. Nitrile‐converting enzymes: an eco‐friendly tool for industrial biocatalysis. Biotechnology and Applied Biochemistry, 60:459-481, Sep 2013. URL: https://doi.org/10.1002/bab.1139, doi:10.1002/bab.1139. This article has 81 citations and is from a peer-reviewed journal.
(ramteke2013nitrile‐convertingenzymesan pages 7-10): Pramod W. Ramteke, Navodita G. Maurice, Babu Joseph, and Bharat J. Wadher. Nitrile‐converting enzymes: an eco‐friendly tool for industrial biocatalysis. Biotechnology and Applied Biochemistry, 60:459-481, Sep 2013. URL: https://doi.org/10.1002/bab.1139, doi:10.1002/bab.1139. This article has 81 citations and is from a peer-reviewed journal.
(moyacuevas2021beyondtheusual pages 1-2): José Moya-Cuevas, Marta-Marina Pérez-Alonso, Paloma Ortiz-García, and Stephan Pollmann. Beyond the usual suspects: physiological roles of the arabidopsis amidase signature (as) superfamily members in plant growth processes and stress responses. Biomolecules, 11:1207, Aug 2021. URL: https://doi.org/10.3390/biom11081207, doi:10.3390/biom11081207. This article has 15 citations.
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(hamilton2020identificationofa pages 3-5): Maureen Hamilton, Andrew L. Consiglio, Kyle MacEwen, A. Joe Shaw, and Vasiliki Tsakraklides. Identification of a yarrowia lipolytica acetamidase and its use as a yeast genetic marker. Microbial Cell Factories, Feb 2020. URL: https://doi.org/10.1186/s12934-020-1292-9, doi:10.1186/s12934-020-1292-9. This article has 13 citations and is from a peer-reviewed journal.
(hamilton2020identificationofa pages 2-3): Maureen Hamilton, Andrew L. Consiglio, Kyle MacEwen, A. Joe Shaw, and Vasiliki Tsakraklides. Identification of a yarrowia lipolytica acetamidase and its use as a yeast genetic marker. Microbial Cell Factories, Feb 2020. URL: https://doi.org/10.1186/s12934-020-1292-9, doi:10.1186/s12934-020-1292-9. This article has 13 citations and is from a peer-reviewed journal.
(ekici2008unconventionalserineproteases pages 11-13): Özlem Doğan Ekici, Mark Paetzel, and Ross E. Dalbey. Unconventional serine proteases: variations on the catalytic ser/his/asp triad configuration. Protein Science, 17:2023-2037, Dec 2008. URL: https://doi.org/10.1110/ps.035436.108, doi:10.1110/ps.035436.108. This article has 445 citations and is from a peer-reviewed journal.
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UniProt: P22580 (AMDY_YEAST) · SGD: S000002650 · Systematic: YDR242W · 549 aa · EC 3.5.1.4 (probable).
Status: dark / understudied gene — no direct biochemical or genetic characterization of the physiological reaction.
The AS (amidase signature) family (Pfam PF01425) is a large, functionally diverse enzyme family whose
members hydrolyse a wide range of carboxamide substrates. They share the Ser-cisSer-Lys catalytic triad
in the conserved "GGSS(G/S)GS" signature region. In P22580 the sequence around residue 209 is
...GGSSGGEGS... (see SQ, "SGGSSGGEGSLIGAHG"), the canonical AS signature block, and the annotated
active-site residues (Ser132, Ser209, Lys — annotated as 233 "acyl-ester intermediate") map onto the
expected triad geometry. This strongly supports assignment to the family and a generic amidase
(carboxamide hydrolase, EC 3.5.1.4) activity, but the AS fold is notoriously substrate-promiscuous
across the family, so fold membership alone does NOT license a specific-substrate MF term.
Conclusion of domain reasoning: a generic "amidase activity" (GO:0004040) MF assignment is
domain-defensible (intact triad + AS fold + Rhea/EC). A specific-substrate amidase term would be
over-annotation and is not supported.
Note: UniProt DR GO also lists GO:0043605 (amide catabolic process, IBA) but this is NOT in the GOA
TSV and the term is OBSOLETE ("unnecessary grouping term"), so it is not reviewed here.
The falcon/Edison report (AMD2-deep-research-falcon.md, 1668 s runtime) reinforces the honest
"dark enzyme, substrate unknown" framing. Load-bearing, independently verified point it surfaced:
Caveats / errors in the falcon report I deliberately did NOT propagate:
- It states AMD2 is 598 aa — WRONG; UniProt P22580 is 549 aa. Not used.
- It asserts a paralog "AMD1 (YCR025C)" — unverified (YCR025C is itself a dubious/uncharacterized
ORF); not asserted in the review.
- It cites cytoplasmic localization from Huh et al. 2003 GFP study — plausible but the paper is not in
cache and I did not verify a usable verbatim quote, so I kept CC as an open knowledge gap (ND retained,
not replaced by a specific CC term). This is the conservative choice for a dark gene.
All AS-family biochemistry statements (Ser-cisSer-Lys triad, generic R-CO-NH2 + H2O -> R-COOH + NH3
reaction, family substrate diversity: FAAH/NAE, plant AMI1/IAM, malonamidase E2, peptide amidase) are
consistent with the UniProt/InterPro record and general enzymology; they are used only as framing, with
the concrete review anchored to UniProt + GOA + PMID:2263500 + PMID:32024536.
id: P22580
gene_symbol: AMD2
product_type: PROTEIN
status: COMPLETE
taxon:
id: NCBITaxon:559292
label: Saccharomyces cerevisiae
description: >-
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, indicating AMD2 is not a
classical acetamidase. It is a non-essential gene of currently uncharacterized
biological role.
references:
- id: GO_REF:0000015
title: Use of the ND evidence code for Gene Ontology (GO) terms
findings: []
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods
findings: []
- id: PMID:2263500
title: Identification of a putative amidase gene in yeast Saccharomyces cerevisiae.
findings: []
reference_review:
relevance: HIGH
correctness: VERIFIED
review_notes: >-
PubMed-verified one-page genomic sequence note (Chang & Abelson, Nucleic
Acids Res 1990). It reports the identification of the YDR242W/AMD2 open
reading frame as a PUTATIVE amidase from sequence similarity only; no
enzyme assay or in-vivo functional characterization is presented.
Abstract-only in cache (full_text_available: false). Supports family
assignment and the "putative/probable" framing, not a specific substrate.
- id: UniProt:P22580
title: AMD2 - Probable amidase - Saccharomyces cerevisiae
findings: []
reference_review:
relevance: HIGH
correctness: VERIFIED
review_notes: >-
UniProtKB reviewed entry (AMDY_YEAST). Domain/family/active-site
annotations (PF01425, IPR023631, PROSITE PS00571, ACT_SITE 132/209/233)
and the EC 3.5.1.4 / Rhea:12020 catalytic activity are used here as the
primary evidence for the generic amidase molecular function. Protein
existence is level 3 (inferred from homology).
- id: PMID:32024536
title: Identification of a Yarrowia lipolytica acetamidase and its use as a yeast genetic marker.
findings: []
reference_review:
relevance: MEDIUM
correctness: VERIFIED
review_notes: >-
PMC full text verified. Not about AMD2 directly, but load-bearing for the
substrate reasoning: S. cerevisiae strain S288C requires a heterologous
acetamidase (A. nidulans amdS or Y. lipolytica YlAMD1) to grow on
acetamide as a nitrogen source, and control transformants (relying only
on the native genome, which includes AMD2) fail to grow on acetamide.
This is direct evidence that AMD2 does not confer classical acetamidase
activity, consistent with AMD2 sitting in a different PANTHER subfamily
(SF11) from the fungal acetamidases (SF9). Supports the "substrate
unknown, not acetamide" MF knowledge gap.
existing_annotations:
- term:
id: GO:0004040
label: amidase activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
qualifier: enables
review:
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.
action: ACCEPT
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.
supported_by:
- reference_id: UniProt:P22580
supporting_text: "Pfam; PF01425; Amidase; 1."
- reference_id: UniProt:P22580
supporting_text: "InterPro; IPR023631; Amidase_dom."
- reference_id: file:yeast/AMD2/AMD2-deep-research-falcon.md
supporting_text: "AMD2 belongs to the Amidase Signature (AS) superfamily"
- term:
id: GO:0003674
label: molecular_function
evidence_type: ND
original_reference_id: GO_REF:0000015
qualifier: enables
review:
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.
action: KEEP_AS_NON_CORE
reason: >-
Uninformative root placeholder retained per GO conventions for ND
annotations; not a curation target and not a core function.
- term:
id: GO:0005575
label: cellular_component
evidence_type: ND
original_reference_id: GO_REF:0000015
qualifier: is_active_in
review:
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).
action: KEEP_AS_NON_CORE
reason: >-
Uninformative root placeholder retained per GO conventions; localization
is genuinely unknown, so no specific CC term can replace it.
- term:
id: GO:0008150
label: biological_process
evidence_type: ND
original_reference_id: GO_REF:0000015
qualifier: involved_in
review:
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.
action: KEEP_AS_NON_CORE
reason: >-
Uninformative root placeholder retained per GO conventions; the in-vivo
biological process is genuinely unknown.
core_functions:
- description: >-
Generic carboxamide hydrolase (amidase) activity inferred from AS-family
membership and an intact Ser-cisSer-Lys catalytic triad. AMD2 is predicted
to hydrolyse an as-yet-unidentified monocarboxylic acid amide substrate to
the corresponding carboxylate and ammonium (EC 3.5.1.4; Rhea:12020). The
substrate specificity, in-vivo reaction, subcellular location, and
biological role are undetermined; only the generic molecular function is
supported.
molecular_function:
id: GO:0004040
label: amidase activity
supported_by:
- reference_id: UniProt:P22580
supporting_text: "Pfam; PF01425; Amidase; 1."
- reference_id: UniProt:P22580
supporting_text: "PROSITE; PS00571; AMIDASES; 1."
- reference_id: UniProt:P22580
supporting_text: "Charge relay system"
knowledge_gaps:
- gap_statement: >-
The physiological substrate and the specific reaction catalysed by
AMD2 in vivo are unknown. EC 3.5.1.4 and Rhea:12020 are generic
parent activities ("a monocarboxylic acid amide"); which carboxamide
AMD2 actually hydrolyses has never been determined experimentally.
boundary: >-
Membership in the amidase signature (AS) family (Pfam PF01425;
InterPro IPR023631; PROSITE PS00571) and retention of the conserved
Ser-cisSer-Lys catalytic triad (UniProt ACT_SITE 132/209/233) reliably
establish a generic carboxamide-hydrolase activity and predict a
catalytically competent enzyme rather than a pseudoenzyme.
gap_kind:
- BIOLOGY
dark_aspect: MF_DARK
status: OPEN
significance: >-
The AS fold is broadly substrate-promiscuous across the family (its
members hydrolyse acetamide, fatty-acid amides, aryl amides, and
peptide-derived amides in different lineages), so fold membership alone
cannot license a specific-substrate term. Identifying the substrate
would convert a generic IEA annotation into a defined, curatable
molecular function.
resolution: >-
Recombinant expression of AMD2 followed by an amidase substrate panel
(e.g. acetamide, longer-chain and aromatic monocarboxamides,
fatty-acid primary amides) with kinetic characterization; metabolomic
comparison of wild-type versus amd2 deletion strains.
provenance:
- reference_id: PMID:2263500
supporting_text: "putative amidase gene"
- reference_id: UniProt:P22580
supporting_text: "RecName: Full=Probable amidase;"
proposed_new_terms: []
suggested_questions:
- question: >-
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?
- question: >-
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?
- question: >-
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?
suggested_experiments:
- hypothesis: >-
AMD2 is a catalytically active amidase whose product feeds nitrogen or
carbon metabolism.
description: >-
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.
experiment_type: enzyme activity assay
- hypothesis: >-
Loss of AMD2 alters a specific amide-containing metabolite pool.
description: >-
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.
experiment_type: comparative metabolomics
- hypothesis: >-
AMD2 localizes to a defined compartment consistent with its metabolic role.
description: >-
Determine AMD2 subcellular localization by C-terminal fluorescent tagging
at the endogenous locus and colocalization with organelle markers, and by
subcellular fractionation.
experiment_type: fluorescence localization
knowledge_gaps:
- gap_statement: >-
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 specifically NOT a classical acetamidase: wild-type
S. cerevisiae S288C (which carries AMD2) cannot grow on acetamide as a
nitrogen source and requires a heterologous acetamidase.
boundary: >-
AS-family membership (Pfam PF01425; InterPro IPR023631; PROSITE PS00571)
and the conserved Ser-cisSer-Lys catalytic triad (UniProt ACT_SITE
132/209/233) firmly establish a generic carboxamide-hydrolase activity
(EC 3.5.1.4; Rhea:12020) and a catalytically competent fold. AMD2 sits in
PANTHER subfamily SF11 ("amidase-related"), distinct from the fungal
acetamidase subfamily SF9.
gap_kind:
- BIOLOGY
dark_aspect: MF_DARK
status: OPEN
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.
resolution: >-
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:
- reference_id: PMID:2263500
supporting_text: "putative amidase gene"
- reference_id: UniProt:P22580
supporting_text: "RecName: Full=Probable amidase;"
- reference_id: PMID:32024536
supporting_text: "Control transformations failed to form colonies while YlAMD1-transformed cells gave rise to well-defined colonies on acetamide plates"
- reference_id: file:interpro/panther/PTHR46072/PTHR46072-entries.csv
supporting_text: "PTHR46072:SF11,AMIDASE-RELATED"
- reference_id: file:interpro/panther/PTHR46072/PTHR46072-entries.csv
supporting_text: "PTHR46072:SF9,ACETAMIDASE"
- gap_statement: >-
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).
boundary: >-
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.
gap_kind:
- BIOLOGY
dark_aspect: BP_DARK
status: OPEN
significance: >-
Placing AMD2 in a metabolic or stress-response pathway would explain the
deletion phenotypes and connect its enzymatic activity to yeast physiology.
resolution: >-
Epistasis/genetic-interaction analysis, condition-specific growth
profiling tied to the identified substrate, and metabolomic pathway
reconstruction.
provenance:
- reference_id: UniProt:P22580
supporting_text: "SIMILARITY: Belongs to the amidase family."
- gap_statement: >-
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.
boundary: >-
UniProt reports no SUBCELLULAR LOCATION for AMD2; the entry annotates only
the amidase domain and catalytic residues, with no targeting features.
gap_kind:
- BIOLOGY
dark_aspect: CC_DARK
status: OPEN
significance: >-
Knowing where AMD2 acts would constrain candidate substrates and its
metabolic context (e.g. cytosolic salvage versus organellar processing).
resolution: >-
Endogenous fluorescent tagging plus organelle-marker colocalization and
subcellular fractionation.
provenance:
- reference_id: UniProt:P22580
supporting_text: "RecName: Full=Probable amidase;"