S. cerevisiae NIT1 = ORF YIL164C, UniProt P40447, SGD S000001426.
199 aa, ~21.6 kDa. UniProt PE=5 ("Uncertain"). This is an UNDERSTUDIED gene: no
gene-specific experimental biochemistry, only sequence-based / high-throughput data.
There are two Nit-domain / carbon-nitrogen-hydrolase paralogs in S. cerevisiae that
are easily confused:
Provenance:
- PMID:28373563 — the characterised yeast enzyme.
- PMID:28373563 — the KO phenotype is for that ortholog (YJL126W per the paper's Table 1 / Methods), not YIL164C.
- SGD curation: yeast NIT2/YJL126W "enables deaminated glutathione amidase activity" (IDA), "hydrolase involved in amide catabolism; localizes to both the cytoplasm and mitochondria" (SGD locus page, WebFetch 2026-07).
- SGD: NIT2 is "one of two proteins in S. cerevisiae with similarity to the Nit domain" — the other is YIL164C (our NIT1).
Implication for this review: the temptation is to transfer the well-characterised
human-NIT1 / yeast-NIT2 dGSH-amidase function onto YIL164C. That would be a
paralog-mis-attribution error. YIL164C's function is genuinely unknown.
The falcon deep-research file fell into exactly this trap (documented, not fixed):
NIT1-deep-research-falcon.md asserts that YIL164C "IS" the dGSH amidase scNit1 and
copies over Peracchi's kinetics, crystal structure and triad. That is incorrect. Two
independent checks show scNit1 is NOT YIL164C:
- Peracchi 2017 Table 1 lists the yeast dGSH-amidase gene as NIT2 and the
ω-amidase gene as NIT3 (WebFetch of PMC5402446, 2026-07); SGD independently
curates NIT2 (YJL126W) with "deaminated glutathione amidase activity" (IDA).
SGD curates YIL164C with ONLY "nitrilase activity (ISA)" — no dGSH amidase, no
experimental MF annotation.
- The scNit1 catalytic-triad numbering in Peracchi/Liu is Glu45-Lys127-Cys169; the
UniProt-predicted triad for YIL164C (P40447) is Glu44-Lys135-Cys169. The Lys
differs (127 vs 135), consistent with scNit1 being a different ORF (NIT2/YJL126W),
not YIL164C. (Cys169 coincidence aside.)
So the falcon report's central functional claim for YIL164C is a paralog conflation.
I retain the falcon file (genuine late artifact) but do NOT use it to support any
functional claim; it is cited only with a reference_review flagging the error.
Yeast nitrilase/Nit-family ORF map (SGD, authoritative):
- NIT1 = YIL164C = P40447 (this gene) — nitrilase-family, function unknown (ISA only).
- NIT2 = YJL126W — deaminated glutathione amidase (IDA); ortholog of human NIT1.
- NIT3 = YLR351C — Nit-domain hydrolase in amide catabolism.
The carbon-nitrogen hydrolase (nitrilase) superfamily is notoriously over-annotated.
Pace & Brenner's foundational classification (PMID:11380987):
Branch 1 (true nitrilases) is the plant IAN→IAA auxin-biosynthesis branch
(Arabidopsis NIT1–4). YIL164C is NOT a plant nitrilase; the SGD-assigned "nitrilase
activity" (ISA, GO:0000257) uses Arabidopsis NIT1 (UniProtKB:P32961) as the
with/from — a genuine branch-1 nitrilase — but transferring plant nitrilase activity
to a divergent fungal paralog is exactly the "haphazard" transfer Pace & Brenner warn
against. Human NIT1 and yeast NIT2 (the characterised orthologs) are amidases, not
nitrilases, so even the closest characterised relatives do not have nitrilase
activity:
PMID:28373563.
UniProt CAUTION for P40447: "Could be the product of a pseudogene. NIT1/YIL164C seems
to be the N-terminal part of a putative nitrilase-like protein formed of NIT1/YIL164C
and YIL165C." (file:yeast/NIT1/NIT1-uniprot.txt)
My own bioinformatics analysis (file:yeast/NIT1/NIT1-bioinformatics/RESULTS.md) supports
and sharpens this:
- The nitrilase E-K-C catalytic triad residues are all present and correctly
positioned in YIL164C: E44 (proton acceptor), K135 (proton donor), C169
(nucleophile, in the diagnostic GGAICWEN motif); they align to the same columns as
the triad of human NIT1, human NIT2 and Arabidopsis NIT1. So YIL164C is NOT a
triad-loss pseudoenzyme.
- BUT YIL164C covers only the N-terminal ~2/3 of the nitrilase fold (alignment
cols 1–270); the adjacent ORF YIL165C (P40446, 119 aa) covers the C-terminal
~1/3 (cols 271–406) with essentially no overlap, and their 318-aa concatenation
spans the full domain (matching human NIT1 327 aa, Arabidopsis 346 aa). So YIL164C
as annotated is a truncated fragment lacking the C-terminal third of the fold
(which contributes to folding/oligomerisation in this superfamily) — unlikely to be
an active enzyme on its own.
- Both YIL164C and YIL165C sit in the uncharacterised PANTHER subfamily
PTHR46044:SF1 "CN hydrolase domain-containing protein", not in any
substrate-defined branch (arylacetonitrilase SF14, cyanide hydratase SF4,
nitrilase-1-related SF11).
SGD independently notes: "in closely related species and other S. cerevisiae strain
backgrounds YIL164C and adjacent ORF, YIL165C, likely constitute a single ORF encoding
a nitrilase gene" (SGD locus page, WebFetch 2026-07); and that YIL164C/YIL165C "have
significant homology to the nitrilase superfamily … but have low sequence homology to
the other two S. cerevisiae nitrilases NIT2 and NIT3" (SGD / Koyama & Walsh 2022
community reference S000316277).
PMID:10959838 (Pace et al. 2000, worm NitFhit crystal structure) is the
original_reference_id for the SGD ISA "nitrilase activity" annotation, but the
with/from field is Arabidopsis NIT1 (P32961). The paper is a structure paper about
C. elegans NitFhit and states the fold has "a presumptive Cys-Glu-Lys catalytic triad"
— it does not demonstrate any substrate for the enzyme, let alone for yeast YIL164C.
Abstract only (full_text_available: false).
KNOWN:
- YIL164C is a verified ORF encoding a carbon-nitrogen-hydrolase-superfamily
(nitrilase-family) domain (Pfam PF00795; PANTHER PTHR46044:SF1).
- It retains an intact, correctly positioned Glu-Lys-Cys catalytic triad (E44/K135/C169).
- It corresponds to the N-terminal ~2/3 of a full nitrilase fold; the C-terminal third
is the adjacent ORF YIL165C; together they equal one full-length domain.
- It is a distinct paralog from yeast NIT2 (YJL126W), which is the characterised dGSH
amidase / ortholog of human NIT1.
- High-throughput deletion phenotypes exist (lifespan, fitness, vacuolar morphology).
NOT KNOWN:
- Its physiological substrate — no substrate has been demonstrated for YIL164C.
- Its catalytic activity in vivo — whether the truncated fragment folds/oligomerises
into an active enzyme at all.
- Its biological process and cellular localization (curated as unknown).
- Whether YIL164C and YIL165C are translated as a single protein in S288C, or whether
YIL164C is a disrupted pseudogene remnant.