NIT1 (YIL164C) is a small (199 aa) putative member of the carbon-nitrogen hydrolase (nitrilase) superfamily in Saccharomyces cerevisiae. It carries a single CN-hydrolase domain (Pfam PF00795) and retains the superfamily's conserved Glu-Lys-Cys catalytic triad (E44, K135, C169). No physiological substrate, catalytic activity, biological process, or subcellular localization has been experimentally established for this protein, and it remains functionally uncharacterized. NIT1 corresponds to only the N-terminal ~2/3 of a complete nitrilase fold; the adjacent ORF YIL165C encodes the complementary C-terminal ~1/3, and in related yeasts the two are a single ORF, so YIL164C as annotated is most likely a truncated fragment of a nitrilase-like protein (UniProt flags it as possibly the product of a pseudogene). It is a distinct paralog from the characterized yeast Nit-domain amidase NIT2 (YJL126W), the ortholog of human NIT1, which hydrolyzes deaminated glutathione; that activity has not been shown for YIL164C. The nitrilase superfamily comprises one branch of true nitrilases and many branches of amidases/amide-condensation enzymes, so family membership alone does not imply nitrilase activity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000257 nitrilase activity | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA-predicted specific nitrilase activity. Pace & Brenner do place the S. cerevisiae NIT1 gene in branch 1 (the true-nitrilase branch), so this is not simply a wrong-branch call; however they describe it as "frequently inactivated", the only demonstrated branch-1 activity is the plant indoleacetonitrile-to-auxin reaction, and no nitrilase substrate has ever been shown for the yeast protein. Combined with the likely truncation (N-terminal 2/3 only), a specific substrate-defining "nitrilase activity" call over-reaches the evidence. Reason: Not wrong at the branch level (branch 1 is the nitrilase branch and yeast NIT1 is placed there), but over-annotated: no nitrilase substrate is demonstrated for this protein, the gene is described as frequently inactivated and appears truncated, and nitrilase-superfamily database annotations are frequently applied without activity data. A specific "nitrilase activity" assertion is stronger than the evidence supports. Supporting Evidence: PMID:11380987 found in plants, animals (C. elegans), fungi (Saccharomyces cerevisiae's frequently inactivated NIT1 gene), and many types of bacteria PMID:11380987 Automated sequence searching easily identifies predicted polypeptides as members of the nitrilase superfamily, but many database annotations have been applied haphazardly. |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Generic InterPro2GO catalytic-activity call. Consistent with the intact catalytic triad but uninformative, and its in-vivo relevance is uncertain given the likely truncation of the fold. Reason: Root-level, non-specific and defensible from family membership plus the conserved triad, but too general to represent a core function and possibly not realized if the protein is a non-functional fragment. Supporting Evidence: file:yeast/NIT1/NIT1-bioinformatics/RESULTS.md All three canonical nitrilase-superfamily triad residues are present at the predicted positions. |
| GO:0016836 hydro-lyase activity | IEA GO_REF:0000117 | REMOVE | Summary: ARBA-predicted hydro-lyase (C-O lyase) activity. This is mechanistically wrong for the nitrilase superfamily, whose members are C-N hydrolases / amidases acting on carbon-nitrogen bonds via the Glu-Lys-Cys triad, not hydro-lyases that cleave carbon-oxygen bonds. Reason: Wrong enzyme class: the carbon-nitrogen hydrolase superfamily hydrolyzes C-N bonds; hydro-lyase (EC 4.2.1.-, C-O lyase) activity is a different catalytic mechanism unsupported by the fold or the conserved triad. This is a spurious ARBA electronic mapping. Supporting Evidence: file:yeast/NIT1/NIT1-uniprot.txt Belongs to the carbon-nitrogen hydrolase superfamily. PMID:11380987 Essentially all members of the nitrilase superfamily have a conserved, apparent catalytic triad of glutamate, lysine and cysteine |
| GO:0000257 nitrilase activity | ISA PMID:10959838 Crystal structure of the worm NitFhit Rosetta Stone protein ... | MARK AS OVER ANNOTATED | Summary: SGD ISA "nitrilase activity" annotation whose with/from is Arabidopsis NIT1 (P32961), a bona fide plant branch-1 nitrilase. Yeast NIT1 is placed in branch 1 by Pace & Brenner, so a nitrilase call is not off-branch; but the cited structure paper (PMID:10959838) establishes only the fold and a presumptive triad, not any substrate, and the only demonstrated branch-1 activity is the plant IAN-to-auxin reaction. No nitrilase substrate is shown for the yeast protein, which is "frequently inactivated" and truncated. Reason: Similarity transfer of a specific substrate-defining activity from a plant nitrilase to an uncharacterized, likely-truncated yeast paralog with no demonstrated substrate. The branch-1 placement makes "nitrilase" plausible at the family level, but asserting the specific activity over-reaches; this is a similarity (ISA) annotation, not experimental. Supporting Evidence: PMID:11380987 found in plants, animals (C. elegans), fungi (Saccharomyces cerevisiae's frequently inactivated NIT1 gene), and many types of bacteria PMID:11380987 Automated sequence searching easily identifies predicted polypeptides as members of the nitrilase superfamily, but many database annotations have been applied haphazardly. |
| GO:0005575 cellular_component | ND GO_REF:0000015 | ACCEPT | Summary: Root cellular_component with ND (no data). No localization has been determined for NIT1 (no GFP datum curated). Reason: Correct use of the ND placeholder given the absence of localization data; flags a genuine knowledge gap. |
| GO:0008150 biological_process | ND GO_REF:0000015 | ACCEPT | Summary: Root biological_process with ND (no data). No biological process has been established for NIT1. Reason: Correct use of the ND placeholder given no process data; flags a genuine knowledge gap. |
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Download this section (compressed HTML)Q: Does NIT1 (YIL164C) have any hydrolase/amidase activity, and on what substrate, when assayed alone versus reconstituted with the YIL165C fragment?
Q: Is YIL164C a functional gene, a truncated fragment, or a pseudogene in S288C, and is a full-length YIL164C+YIL165C protein ever translated?
Q: Does NIT1 have a biological role distinct from the paralogous dGSH-amidase NIT2 (YJL126W), or is it functionally inert/redundant?
Experiment: Express and purify (i) YIL164C alone and (ii) the YIL164C+YIL165C fusion, and screen both against nitrile and amide substrate panels (including deaminated glutathione and Ξ±-ketoglutaramate as paralog controls), measuring kinetics and confirming triad dependence with active-site mutants.
Hypothesis: NIT1 retains the ancestral C-N hydrolase/amidase activity but only as the reconstituted full-length YIL164C+YIL165C protein.
Type: recombinant enzyme substrate profiling
Experiment: Compare untargeted metabolomes of nit1-delta, nit2-delta, nit3-delta and double mutants under standard and starvation/anaerobic conditions to detect a NIT1-specific accumulating substrate.
Hypothesis: NIT1 loss perturbs a specific metabolite pool distinct from that of NIT2.
Type: comparative metabolomics of deletion strains
Experiment: Use ribosome profiling and targeted proteomics to detect the YIL164C product and determine whether translation extends into YIL165C.
Hypothesis: YIL164C is translated (functional ORF) rather than being a silent pseudogene.
Type: ribosome profiling / targeted proteomics
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The physiological substrate and catalytic activity of NIT1 (YIL164C) are unknown. It has never been shown to hydrolyze a nitrile or any amide/other C-N bond in vitro or in vivo, and it is unresolved whether the protein is catalytically active at all.
OPEN BIOLOGY MF_DARK
What is known: Established: NIT1 is a carbon-nitrogen hydrolase (nitrilase) superfamily member with a single CN-hydrolase domain and an intact, correctly positioned Glu-Lys-Cys catalytic triad (E44/K135/C169); Pace & Brenner place the yeast NIT1 gene in branch 1 (the true-nitrilase branch, EC 3.5.5.1) but call it "frequently inactivated". Unknown: which C-N bond it acts on (if any) and whether catalytic competence is realized in this protein. Even within branch 1 the only demonstrated activity is the plant indoleacetonitrile-to-auxin reaction, so branch placement does not fix a substrate for the yeast protein.
Significance: NIT1 is a conserved-family enzyme candidate with no assigned function; it is also an archetype of nitrilase-superfamily over-annotation, where generic "nitrilase activity" is asserted without a demonstrated substrate.
What would resolve it: Biochemical substrate profiling of recombinant protein (nitrile and amide panels, including deaminated glutathione and omega-amidase substrates as negative/positive controls), and metabolomic comparison of a clean deletion strain.
Provenance (the field's own admissions):
Gap: It is unresolved whether YIL164C is a functional gene or a truncated/pseudogene fragment, and whether YIL164C and YIL165C are translated as one protein in S. cerevisiae S288C.
OPEN BIOLOGYCURATION MF_DARK
What is known: Established: YIL164C encodes the N-terminal ~2/3 of a nitrilase fold and YIL165C the C-terminal ~1/3; together they equal a full-length domain, and in related yeasts/strains they form a single ORF. UniProt lists PE=5 (Uncertain) and flags possible pseudogene status. Unknown: the actual translated product in S288C and whether a functional full-length enzyme is ever made.
Significance: Determines whether NIT1 should be curated as a functional enzyme, a fragment, or a pseudogene, and whether activity assays should use YIL164C alone or the YIL164C+YIL165C concatenation.
What would resolve it: Ribosome-profiling / proteomics to establish the translated product(s), and activity testing of the reconstituted full-length YIL164C+YIL165C protein versus YIL164C alone.
Provenance (the field's own admissions):
Gap: The biological process and subcellular localization of NIT1 are unknown; its high-throughput deletion phenotypes have no established mechanistic link to a molecular function.
OPEN BIOLOGY WHOLLY_DARK
What is known: Established: SGD curates BP and CC as unknown (ND); deletion screens report pleiotropic phenotypes. Unknown: the pathway NIT1 acts in and where in the cell it acts. The characterized dGSH-amidase / metabolite-repair role belongs to the paralog NIT2 (YJL126W), not to YIL164C.
Significance: Without a process or localization, NIT1 cannot be placed in any pathway or GO-CAM; distinguishing its role from the paralogous NIT2 amidase is required to avoid mis-attribution.
What would resolve it: GFP localization, targeted metabolomics of the deletion, and epistasis with NIT2/NIT3 to test for a distinct (non-redundant) role.
Provenance (the field's own admissions):
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