Affinage

NIT2

Omega-amidase NIT2 · UniProt Q9NQR4

Length
276 aa
Mass
30.6 kDa
Annotated
2026-06-10
41 papers in source corpus 10 papers cited in narrative 10 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/5 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

NIT2 is a ubiquitously expressed cytosolic omega-amidase (EC 3.5.1.3) that hydrolyzes alpha-ketoglutaramate and alpha-ketosuccinamate—the alpha-keto acid analogues of glutamine and asparagine—to alpha-ketoglutarate and oxaloacetate, thereby channeling the products of transamination reactions into the TCA cycle (PMID:19595734, PMID:19596042, PMID:19464248). Recombinant reconstitution established that this activity belongs specifically to NIT2 (nitrilase 2) rather than NIT1 (PMID:19596042), and the catalysis proceeds through a Cys-Glu-Lys triad (C153, E43, K112 in human numbering), with mutation of any triad residue abolishing turnover (PMID:22674578). The catalytic cysteine is redox-sensitive: H2O2-mediated oxidation reversibly inhibits NIT2, and endothelial-specific loss of NIT2 raises alpha-ketoglutaramate and impairs angiogenesis, proliferation, and sprouting while inducing senescence, positioning NIT2 as a redox-gated link between glutamine catabolism and endothelial function [PMID:bio_10.1101_2024.08.28.610061]. Beyond metabolism, NIT2 has a non-enzymatic moonlighting role in which it binds BRD1 to suppress HBO1-mediated H3K14 acetylation and RELA-driven oxidative phosphorylation gene expression; Src phosphorylation of NIT2 at Y49 drives its dissociation from BRD1, CCNB1IP1-dependent autophagic degradation, and consequent chemoresistance in gastric cancer (PMID:39565874). NIT2 expression is post-transcriptionally controlled by NAT10-mediated ac4C modification that stabilizes its mRNA and enhances glutamine metabolism in lung cancer cells (PMID:42030693). Early overexpression studies also link NIT2 to G2 cell-cycle arrest accompanied by altered 14-3-3 isoform expression (PMID:17488281).

Mechanistic history

Synthesis pass · year-by-year structured walk · 8 steps
  1. 2009 High

    Established the molecular identity and biochemical activity of mammalian NIT2 by showing it is the cytosolic omega-amidase that hydrolyzes the alpha-keto acid analogues of glutamine and asparagine.

    Evidence Biochemical purification (~3600-fold) from rat liver cytosol with parallel alpha-ketoglutaramate hydrolysis and succinamate hydroxaminolysis assays, plus recombinant E. coli expression distinguishing Nit2 from Nit1

    PMID:19464248 PMID:19595734 PMID:19596042

    Open questions at the time
    • Did not define the active-site residues or catalytic mechanism
    • Physiological flux contribution to TCA replenishment not quantified in vivo
  2. 2007 Medium

    Connected NIT2 to a cellular growth phenotype, showing ectopic expression arrests cells in G2 with altered 14-3-3 isoform levels.

    Evidence Subcellular fractionation, ectopic overexpression in HeLa cells, flow cytometry, proteomics and RT-PCR

    PMID:17488281

    Open questions at the time
    • No mechanistic reconstitution linking enzyme activity to G2 arrest
    • Single lab, overexpression-based; endogenous role untested
  3. 2012 High

    Defined the catalytic machinery of human NIT2 as a Cys-Glu-Lys triad and identified a substrate-binding loop, explaining substrate specificity.

    Evidence Site-directed mutagenesis (E43A, K112A, C153A) with kinetic assays plus molecular dynamics simulation

    PMID:22674578

    Open questions at the time
    • No experimental crystal structure of human NIT2
    • Order of chemical steps not resolved by these data
  4. 2013 Medium

    Provided structural snapshots of reaction intermediates covalently bound to the catalytic cysteine, informing the Nit subfamily mechanism.

    Evidence X-ray crystallography of wild-type and C169S yeast Nit2 enzyme-intermediate complexes with activity assays

    PMID:23897470

    Open questions at the time
    • Performed on yeast Nit2, a homolog of mammalian Nit1, not mammalian NIT2
    • alpha-ketoglutaramate is a poor substrate for the yeast enzyme, limiting direct extrapolation
  5. 2021 Low

    Resolved the stepwise catalytic mechanism, identifying the rate-limiting tetrahedral intermediate and the catalytic roles of Cys191 and Glu81.

    Evidence QM/MM computational simulation validated against experimental kcat

    PMID:33463886

    Open questions at the time
    • Computational only; no new wet-lab validation of the proposed intermediates
    • Does not address regulation of the enzyme in cells
  6. 2024 Medium

    Revealed NIT2 as a redox-sensitive metabolic switch whose oxidative inactivation and loss impair endothelial angiogenesis via alpha-ketoglutaramate accumulation.

    Evidence Endothelial-specific NIT2 knockout mice, metabolomic alpha-ketoglutaramate measurement, H2O2 activity inhibition, sprouting/proliferation assays (preprint)

    PMID:bio_10.1101_2024.08.28.610061

    Open questions at the time
    • Preprint, single lab, not peer-reviewed
    • Specific oxidized cysteine residue not definitively mapped
    • Mechanism linking alpha-ketoglutaramate to senescence unresolved
  7. 2024 Medium

    Uncovered a non-enzymatic moonlighting function of NIT2 in chromatin and transcriptional control, linking its Src-driven degradation to chemoresistance.

    Evidence CRISPR-Cas9 screen, Co-IP, patient-derived organoids, xenografts, phosphorylation mapping, phase separation and ubiquitination assays

    PMID:39565874

    Open questions at the time
    • Mechanistic complexity from a single lab, not independently replicated
    • Structural basis of NIT2-BRD1 interaction unknown
    • Generality beyond gastric cancer untested
  8. 2026 Medium

    Identified post-transcriptional control of NIT2 by NAT10-mediated ac4C mRNA modification driving glutamine metabolism in cancer.

    Evidence meRIP, dual-luciferase reporter, RNAi, overexpression rescue, xenograft, metabolic assays

    PMID:42030693

    Open questions at the time
    • Specific ac4C sites on NIT2 mRNA not mapped
    • Single lab; relationship to the moonlighting and redox functions unexplored

Open questions

Synthesis pass · forward-looking unresolved questions
  • How NIT2's metabolic, redox-switch, and chromatin-regulatory functions are integrated within a single cell, and whether they are coordinately regulated, remains unresolved.
  • No unified model connecting omega-amidase activity to BRD1-dependent transcriptional control
  • No experimental structure of human NIT2 or the NIT2-BRD1 complex

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016787 hydrolase activity 4 GO:0016740 transferase activity 2 GO:0140299 molecular sensor activity 1
Localization
GO:0005829 cytosol 2
Pathway
R-HSA-1430728 Metabolism 3 R-HSA-4839726 Chromatin organization 1
Partners
BRD1CCNB1IP1SRC

Evidence

Reading pass · 10 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2009 Rat liver Nit2 was identified as omega-amidodicarboxylate amidohydrolase (omega-amidase, EC 3.5.1.3), catalyzing hydrolysis of alpha-ketoglutaramate (the alpha-keto acid analogue of glutamine) and alpha-ketosuccinamate (the alpha-keto acid analogue of asparagine) to alpha-ketoglutarate and oxaloacetate, respectively; the enzyme is located in the cytosol and is ubiquitously expressed. Biochemical purification from rat liver cytosol, enzymatic assay with identified substrates (alpha-ketoglutaramate and alpha-ketosuccinamate), co-purification tracking Biochimie High 19595734 19596042
2009 Mouse nitrilase 2 (Nit2) expressed in E. coli and purified was shown to catalyze hydrolysis of alpha-ketoglutaramate and other known omega-amidase substrates; mouse nitrilase 1 showed no such activity, establishing Nit2 (not Nit1) as the mammalian omega-amidase. Recombinant protein expression in E. coli, enzymatic activity assay with alpha-ketoglutaramate substrate Biochimie High 19596042
2009 Purification of omega-amidase approximately 3600-fold from rat liver cytosol using alpha-ketoglutaramate hydrolysis and succinamate hydroxaminolysis assays confirmed the enzyme's activity toward both substrates, with the ratio of activities remaining constant throughout purification, establishing these as substrates of the same enzyme (Nit2). Multi-step biochemical purification (~3600-fold) from rat liver cytosol, two independent enzymatic activity assays (alpha-ketoglutaramate hydrolysis and succinamate hydroxaminolysis) tracked in parallel Analytical biochemistry High 19464248
2007 Human NIT2 protein is distributed mainly in the cytosol; ectopic overexpression of Nit2 in HeLa cells inhibits cell growth through G2 arrest (not apoptosis); Nit2 overexpression up-regulates 14-3-3sigma (an inhibitor of G2/M progression and Akt-activated growth) and down-regulates 14-3-3beta. Subcellular fractionation, ectopic overexpression in HeLa cells, flow cytometric cell cycle analysis, proteomic and RT-PCR analyses of downstream effectors The FEBS journal Medium 17488281
2012 Human Nit2/omega-amidase has a catalytic triad of E43, K112, and C153; site-directed mutagenesis of each residue (E43A, K112A, C153A) impaired catalytic activity; deletion of loop 116-128 also disrupted substrate binding and turnover; molecular dynamics simulations confirmed the role of these residues in substrate specificity toward alpha-ketoglutaramate and succinamate. Site-directed mutagenesis of active-site residues, kinetic activity assays with alpha-ketoglutaramate and succinamate substrates, molecular dynamics simulation The Journal of biological chemistry High 22674578
2013 Crystal structures of yeast Nit2 (a homolog of mammalian Nit1, not Nit2) in complex with alpha-ketoglutarate and oxaloacetate revealed that these products are covalently bound to the catalytic Cys169 via a thioester bond, reflecting reaction intermediates; this provided structural insights into the catalytic mechanism of the Nit subfamily. Notably, alpha-ketoglutaramate is a relatively poor substrate for yeast Nit2, distinguishing it from mammalian Nit2. X-ray crystallography of wild-type and C169S mutant enzyme-intermediate complexes, enzymatic activity assay Acta crystallographica. Section D, Biological crystallography Medium 23897470
2021 QM/MM computational study of Nit2 established the catalytic mechanism involves four steps: (1) nucleophilic attack of Cys191 on alpha-ketosuccinamate, (2) formation of a first tetrahedral intermediate (rate-limiting step, calculated barrier 18.4 kcal/mol consistent with experimental kcat), (3) formation of a second tetrahedral intermediate, and (4) hydrolysis of a thioacyl-enzyme intermediate to release oxaloacetate; Cys191 and Glu81 play active catalytic roles while Lys150 plays a secondary role. QM/MM computational simulation validated against experimental kcat values Chemphyschem Low 33463886
2024 NIT2 interacts physically with bromodomain-containing protein BRD1 to inhibit HBO1-mediated acetylation of histone H3 at lysine-14 (H3K14ac) and suppress RELA-targeted oxidative phosphorylation (OXPHOS) gene expression, independent of its metabolic (omega-amidase) function; Src kinase phosphorylates NIT2 at Y49, promoting NIT2 dissociation from BRD1 and subsequent binding to E3 ligase CCNB1IP1, leading to autophagic degradation of NIT2; reduced NIT2 protein levels allow BRD1 to form phase separation and increase H3K14ac, and also stabilize RELA by suppressing ING4-mediated RELA ubiquitination. CRISPR-Cas9 screen, Co-IP, patient-derived organoids, xenograft tumors, phosphorylation assay, phase separation assay, ubiquitination assay Science translational medicine Medium 39565874
2024 Reversible oxidation of specific cysteine residue(s) in NIT2 by H2O2 inhibits its catalytic activity; endothelial-specific knockout of NIT2 in mice leads to increased alpha-ketoglutaramate (αKGM) levels and impaired angiogenesis, with knockout cells showing impaired proliferation, sprouting, and induced senescence, establishing NIT2 as a redox-sensitive enzyme linking glutamine catabolism to endothelial function. Endothelial-specific NIT2 knockout mice, metabolomic measurement of αKGM, H2O2 treatment with activity assay, endothelial cell sprouting and proliferation assays bioRxivpreprint Medium bio_10.1101_2024.08.28.610061
2026 NAT10 (the only known ac4C transferase) promotes NIT2 mRNA stability through ac4C modification, increasing NIT2 expression and thereby enhancing glutamine metabolism (glutamine consumption, alpha-ketoglutarate and ATP production) in lung cancer cells; overexpression of NIT2 rescued the suppression of cell viability, proliferation, migration, and glutamine metabolism caused by NAT10 knockdown. Methylated RNA immunoprecipitation (meRIP), dual-luciferase reporter assay, RNA interference, overexpression rescue, xenograft model, metabolic assays Molecular immunology Medium 42030693

Source papers

Stage 0 corpus · 41 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1990 nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain. Molecular and cellular biology 189 2137552
1990 nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein. Proceedings of the National Academy of Sciences of the United States of America 188 2142530
1993 Isolation of the Chlamydomonas regulatory gene NIT2 by transposon tagging. Genetics 128 8394263
2007 Nitrate signaling by the regulatory gene NIT2 in Chlamydomonas. The Plant cell 96 18024571
1987 Characterization of nit-2, the major nitrogen regulatory gene of Neurospora crassa. Molecular and cellular biology 90 2885741
1995 The negative-acting NMR regulatory protein of Neurospora crassa binds to and inhibits the DNA-binding activity of the positive-acting nitrogen regulatory protein NIT2. Biochemistry 70 7612627
2003 CLNR1, the AREA/NIT2-like global nitrogen regulator of the plant fungal pathogen Colletotrichum lindemuthianum is required for the infection cycle. Molecular microbiology 69 12694611
1987 Complementation of areA- regulatory gene mutations of Aspergillus nidulans by the heterologous regulatory gene nit-2 of Neurospora crassa. Proceedings of the National Academy of Sciences of the United States of America 54 2954160
2009 Identification of the putative tumor suppressor Nit2 as omega-amidase, an enzyme metabolically linked to glutamine and asparagine transamination. Biochimie 49 19595734
2005 Ammonium transporter genes in Chlamydomonas: the nitrate-specific regulatory gene Nit2 is involved in Amt1;1 expression. Plant molecular biology 47 15821986
1994 DNA recognition by the NIT2 nitrogen regulatory protein: importance of the number, spacing, and orientation of GATA core elements and their flanking sequences upon NIT2 binding. Biochemistry 46 8286388
1992 Expression of chimeric genes by the light-regulated cabII-1 promoter in Chlamydomonas reinhardtii: a cabII-1/nit1 gene functions as a dominant selectable marker in a nit1- nit2- strain. Molecular and cellular biology 46 1406696
2009 Molecular identification of omega-amidase, the enzyme that is functionally coupled with glutamine transaminases, as the putative tumor suppressor Nit2. Biochimie 42 19596042
2007 Growth inhibitory effect of the human NIT2 gene and its allelic imbalance in cancers. The FEBS journal 42 17488281
1997 Two distinct protein-protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa. Molecular microbiology 39 9427402
1993 Recognition of specific nucleotide bases and cooperative DNA binding by the trans-acting nitrogen regulatory protein NIT2 of Neurospora crassa. Nucleic acids research 32 8396761
1992 NIT2, the nitrogen regulatory protein of Neurospora crassa, binds upstream of nia, the tomato nitrate reductase gene, in vitro. Current genetics 32 1531184
2009 Assay and purification of omega-amidase/Nit2, a ubiquitously expressed putative tumor suppressor, that catalyzes the deamidation of the alpha-keto acid analogues of glutamine and asparagine. Analytical biochemistry 31 19464248
1986 Molecular cloning of nit-2, a regulatory gene required for nitrogen metabolite repression in Neurospora crassa. Gene 31 2879771
2015 Downregulation of NIT2 inhibits colon cancer cell proliferation and induces cell cycle arrest through the caspase-3 and PARP pathways. International journal of molecular medicine 26 25738796
1999 The NIT2 nitrogen regulatory protein of Neurospora: expression and stability of nit-2 mRNA and protein. Current genetics 26 10501938
1990 Site-directed mutagenesis of the 'zinc finger' DNA-binding domain of the nitrogen-regulatory protein NIT2 of Neurospora. Molecular microbiology 21 2150539
2024 NIT2 dampens BRD1 phase separation and restrains oxidative phosphorylation to enhance chemosensitivity in gastric cancer. Science translational medicine 19 39565874
1979 Regulation of glutamate dehydrogenases in nit-2 and am mutants of Neurospora crassa. Journal of bacteriology 19 35517
1995 Binding affinity and functional significance of NIT2 and NIT4 binding sites in the promoter of the highly regulated nit-3 gene, which encodes nitrate reductase in Neurospora crassa. Journal of bacteriology 17 7592372
2013 Structures of enzyme-intermediate complexes of yeast Nit2: insights into its catalytic mechanism and different substrate specificity compared with mammalian Nit2. Acta crystallographica. Section D, Biological crystallography 16 23897470
1994 DNA binding site specificity of the Neurospora global nitrogen regulatory protein NIT2: analysis with mutated binding sites. Molecular & general genetics : MGG 15 7808401
1993 Amino-acid substitutions in the zinc finger of NIT2, the nitrogen regulatory protein of Neurospora crassa, alter promoter element recognition. Current genetics 15 8221929
2012 Structural insights into the catalytic active site and activity of human Nit2/ω-amidase: kinetic assay and molecular dynamics simulation. The Journal of biological chemistry 13 22674578
2014 Chlamydomonas NZF1, a tandem-repeated zinc finger factor involved in nitrate signalling by controlling the regulatory gene NIT2. Plant, cell & environment 10 24548141
2017 Nuclear transport of the Neurospora crassa NIT-2 transcription factor is mediated by importin-α. The Biochemical journal 9 29054975
1983 Trans-nuclear action of the nit-2 regulatory gene product and study of two additional nitrogen control genes in Neurospora crassa. Current genetics 9 24173118
2021 An Unsual Cys-Glu-Lys Catalytic Triad is Responsible for the Catalytic Mechanism of the Nitrilase Superfamily: A QM/MM Study on Nit2. Chemphyschem : a European journal of chemical physics and physical chemistry 8 33463886
1996 Identification of the native NIT2 major nitrogen regulatory protein in nuclear extracts of Neurospora crassa. Genetica 8 8901135
1988 Xanthine dehydrogenase expression in Neurospora crassa does not require a functional nit-2 regulatory gene. Biochemical genetics 6 2967694
1993 Regulation of ammonium ion assimilation enzymes in Neurospora crassa nit-2 and ms-5 mutant strains. Biochemical genetics 5 7907211
1992 The regulatory gene nit-2 of Neurospora crassa complements a nnu mutant of Gibberella zeae (Fusarium graminearum). Molecular & general genetics : MGG 4 1465117
2024 Biodegradation of nitenpyram (neonicotinoid insecticide) by endophytic bacterium, Bacillus thuringiensis strain NIT-2, isolated from neonicotinoid-treated plant samples. Journal of pesticide science 3 38882709
2021 A novel efficient producer of human ω-amidase (Nit2) in Escherichia coli. Analytical biochemistry 2 34391728
2025 Ustilago maydis Nit2 Regulates Nitrate Utilisation During Biotrophy and Affects Amino Acid Metabolism of Galls Under Nitrogen Depletion. Molecular plant pathology 1 40891006
2026 NAT10 promotes lung cancer progression by enhancing glutamine metabolism through increasing ac4C modification on NIT2. Molecular immunology 0 42030693

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