Affinage

GART

Trifunctional purine biosynthetic protein adenosine-3 · UniProt P22102

Length
1010 aa
Mass
107.8 kDa
Annotated
2026-06-10
40 papers in source corpus 16 papers cited in narrative 15 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

GART encodes a trifunctional enzyme catalyzing sequential steps of de novo purine (IMP) biosynthesis, with a single human gene complementing GARS, AIRS, and GART enzymatic deficiencies in mutant cells (PMID:2050105). The same locus also yields a monofunctional ~50 kDa GARS protein through alternative splicing using an intronic polyadenylation site, and both products are developmentally regulated in cerebellum (PMID:9328467). Structurally, the full-length protein dimerizes through its middle domain in an approximate seesaw geometry with mobile terminal enzyme units, a configuration consistent with internal substrate/product transfer (PMID:20631005); catalysis at the formyltransferase site depends on His108 acting as an acid catalyst paired with Asp144 and a supporting histidine network, while Glu75 and Gly684 are essential for the GARS and AIRS activities respectively (PMID:11604543, PMID:19007868). Genetic loss of function dissects the pathway physiologically: in zebrafish, GTP-pathway deficiency produces pigmentation defects and ATP-pathway deficiency causes microphthalmia via failure of retinoblast cell-cycle exit (PMID:19570845). Beyond its canonical metabolic role, GART carries a non-canonical methyltransferase activity centered on E948 that methylates RUVBL1 at K7 to stabilize it and activate Wnt/β-catenin signaling, promoting tumor stemness in colorectal cancer (PMID:37439412), and under glucose limitation AMPK phosphorylates GART at Ser440 to drive UCK2 binding and THF-dependent UCK2 stabilization, supporting pyrimidine salvage for tumor growth (PMID:39865175). In Drosophila, peripheral Gart is directly regulated by the CLOCK/CYCLE heterodimer through E-box elements and integrated into an NPF/NPFR feedback loop, linking purine synthesis to circadian feeding control (PMID:37531254, PMID:42188194).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 1991 High

    Whether a single gene accounts for all three early purine-pathway activities was unresolved; demonstrating that one transferred gene complements GARS- and GARS/AIRS-deficient cells established GART as a single trifunctional locus.

    Evidence YAC DNA transfer into mutant CHO cells with complementation of purine auxotrophy

    PMID:2050105

    Open questions at the time
    • Did not define the protein's domain architecture or catalytic residues
    • No structural information on the trifunctional polypeptide
  2. 1997 High

    It was unknown whether the locus produced more than the trifunctional enzyme; antibody detection showed a monofunctional 50 kDa GARS isoform arises by alternative splicing via an intronic polyadenylation site, revealing isoform-level regulation.

    Evidence Domain-specific antibody Western blot in transfected CHO cells plus developmental expression analysis

    PMID:9328467

    Open questions at the time
    • Functional distinction between monofunctional and trifunctional products not established
    • Tissue-specific regulation of splicing unresolved
  3. 2001 Medium

    The catalytic chemistry of the formyltransferase site was undefined; electrostatic analysis of a ternary complex identified His108 as an acid catalyst coupled to Asp144 with GAR as nucleophile, defining the proton-transfer mechanism.

    Evidence Poisson-Boltzmann electrostatic calculations on the crystallographic GART ternary complex

    PMID:11604543

    Open questions at the time
    • Predicted pKa values not experimentally validated by mutagenesis or kinetics
    • Computational rather than direct biochemical demonstration
  4. 2003 Low

    The structural basis of catalytic-site stability was unclear; continuum electrostatic modeling quantified a five-histidine/two-aspartate network as the dominant contributor to active-site stability.

    Evidence Continuum electrostatic calculations and structural modeling of activation helix stability

    PMID:14499900

    Open questions at the time
    • Computational predictions only, no experimental validation
    • Functional consequence of disrupting individual histidines untested
  5. 2008 Medium

    Residue-level requirements for the GARS and AIRS activities were undefined; mutagenesis with auxotrophy selection identified Glu75 and Gly684 as essential for the respective activities.

    Evidence Site-directed mutagenesis in CHO-K1 cells with purine auxotrophy selection

    PMID:19007868

    Open questions at the time
    • Single lab, no kinetic characterization of mutants
    • Structural basis of essentiality not resolved
  6. 2008 High

    The physiological consequences of GART loss were unknown; zebrafish loss-of-function dissected pigmentation defects (GTP pathway) from microphthalmia driven by failed retinoblast cell-cycle exit (ATP pathway).

    Evidence Zebrafish recessive mutant analysis with purine-metabolite rescue experiments

    PMID:19570845

    Open questions at the time
    • Does not address mammalian developmental requirements
    • Molecular link between purine pools and cell-cycle exit not detailed
  7. 2010 High

    The quaternary organization of the full-length enzyme was unknown; combined crystallography and SAXS revealed a middle-domain dimer with mobile terminal units in a seesaw geometry permitting possible substrate channeling.

    Evidence X-ray crystallography of GARS and AIRS domains plus SAXS of full-length protein

    PMID:20631005

    Open questions at the time
    • Direct demonstration of substrate channeling not shown
    • Full-length structure resolved only at low SAXS resolution
  8. 2016 Low

    Whether GART has roles beyond metabolic supply was unexplored; knockdown in intestinal epithelial cells linked GART loss to apoptosis and impaired migration via MEKK3-MKK3-p38/p53/PUMA, framing it as a barrier-integrity regulator.

    Evidence siRNA knockdown in cultured IECs with apoptosis, migration, and pathway Western blots

    PMID:27718035

    Open questions at the time
    • Pathway placement by knockdown and Western blot without direct mechanistic reconstitution
    • Whether the effect is enzymatic or non-canonical unresolved
  9. 2023 Medium

    A non-canonical enzymatic function was discovered; GART acts as a methyltransferase via E948 that methylates RUVBL1-K7 to stabilize it and activate Wnt/β-catenin, promoting colorectal cancer stemness.

    Evidence In vitro methyltransferase assay, E948 mutagenesis, Co-IP, and xenograft/PDX models

    PMID:37439412

    Open questions at the time
    • Novel methyltransferase activity awaits independent replication
    • Structural basis for substrate recognition by E948 site not defined
  10. 2023 Medium

    Whether GART couples to hormone-driven proliferation was unclear; knockdown or lometrexol drives ERα degradation and blocks proliferation downstream of VGLL3 induction, rescuable by IMP, tying GART's metabolic output to cancer growth.

    Evidence siRNA screen, metabolomics, VGLL3 overexpression, lometrexol inhibition, and IMP rescue across two labs

    PMID:35434822 PMID:37143728

    Open questions at the time
    • Mechanism linking IMP supply to ERα stability not defined
    • Direct molecular partners not identified
  11. 2023 Medium

    Whether purine synthesis is wired to circadian physiology was unknown; in Drosophila peripheral Gart is directly transcriptionally controlled by CLOCK/CYCLE via E-boxes and regulates feeding rhythms and energy homeostasis in a tissue-specific manner.

    Evidence Tissue-specific Gart knockdown, E-box reporter assays, behavioral feeding assays, and genetic epistasis

    PMID:37531254

    Open questions at the time
    • Conservation of circadian regulation in mammals not addressed
    • Metabolic output linking Gart to feeding behavior not detailed
  12. 2025 Medium

    The signaling context of GART beyond metabolism expanded; under glucose limitation AMPK phosphorylates GART-Ser440 to drive UCK2 binding and THF-dependent UCK2 stabilization, sustaining pyrimidine salvage, while separate work placed GART upstream of HSP90α/CDK6 and PAICS-Akt-β-catenin proliferative axes.

    Evidence Co-IP, phospho-specific assays, AMPK reconstitution, siRNA, and xenograft models

    PMID:39865175 PMID:40185325 PMID:40201340

    Open questions at the time
    • Whether moonlighting roles require enzymatic versus scaffold function varies and is unresolved
    • PAICS-axis placement based on Western blot without direct interaction evidence
  13. 2026 Medium

    The systemic regulation of Gart in feeding was extended; NPF/NPFR signaling positively regulates intestinal Gart with reciprocal negative feedback on NPF, forming a peripheral feeding-control loop with Gart acting downstream of NPF.

    Evidence Genetic epistasis, tissue-specific knockdown, NPF receptor manipulation, and feeding assays in Drosophila

    PMID:42188194

    Open questions at the time
    • Molecular basis of Gart's feedback on NPF expression unknown
    • Mammalian relevance untested

Open questions

Synthesis pass · forward-looking unresolved questions
  • How the canonical trifunctional enzyme is mechanistically reconciled with its diverse non-canonical activities (methyltransferase, AMPK-regulated scaffold, Wnt/β-catenin and proliferative signaling) within a single protein remains unresolved.
  • No structure of the E948 methyltransferase site or its substrate engagement
  • Whether moonlighting functions depend on the trifunctional versus monofunctional isoform is unknown
  • Direct in vivo demonstration that purine flux versus protein-binding drives each phenotype lacking

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016740 transferase activity 2 GO:0016874 ligase activity 2 GO:0140657 ATP-dependent activity 2 GO:0140096 catalytic activity, acting on a protein 1
Pathway
R-HSA-162582 Signal Transduction 3 R-HSA-1430728 Metabolism 2 R-HSA-9909396 Circadian clock 2
Partners
HSP90AA1RUVBL1UCK2

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2010 Crystal structures of two domains of human trifunctional GART were solved: the glycinamide ribonucleotide synthetase (GARS) domain and the aminoimidazole ribonucleotide synthetase (AIRS) domain. Small-angle X-ray scattering models of the full-length protein indicate it forms a dimer through the middle domain, with an approximate seesaw geometry where terminal enzyme units display high mobility owing to flexible linker segments, potentially facilitating internal substrate/product transfer. X-ray crystallography of individual domains; small-angle X-ray scattering (SAXS) of full-length protein Nucleic acids research High 20631005
2001 Electrostatic calculations using the crystal structure of a GART ternary complex (with pseudosubstrate 5-deaza-5,6,7,8-THF and substrate GAR) showed strong electrostatic coupling among active-site residues His108, Asp144, and substrate GAR. His108 acts as an acid catalyst (forming a salt bridge with Asp144) and GAR acts as a nucleophile, with His137 also providing critical three-way electrostatic stabilization of the catalytic site. Proton exchange between GAR and His108 is geometry- and distance-dependent. Poisson-Boltzmann electrostatic calculations on crystallographic ternary complex; theoretical mutagenesis and deletion constructs Protein science Medium 11604543
2003 Continuum electrostatic calculations on GART revealed that a network of five histidines (His108, His119, His121, His132, His137) and two aspartic acids (Asp141, Asp144) contributes ~12 kcal/mol of the ~16 kcal/mol total stability of the catalytic site, with the His121-His132 interaction contributing ~2.2 kcal/mol to ionization free energy, demonstrating the importance of this histidine network for catalytic-site stability. Continuum electrostatic calculations and structural modeling of activation helix stability Biophysical chemistry Low 14499900
1997 The human GARS-AIRS-GART gene encodes not only the trifunctional 110 kDa protein but also a monofunctional 50 kDa GARS protein produced by alternative splicing that uses a polyadenylation site in the intron between the terminal GARS and first AIRS exons. Both proteins are developmentally regulated in human cerebellum. Western blot with domain-specific monoclonal and polyclonal antibodies on CHO cells transfected with human GARS-AIRS-GART gene; developmental expression analysis Human molecular genetics High 9328467
2008 Loss-of-function mutations in zebrafish gart (encoding the trifunctional GARS/AIRS/GART enzyme) result in pigmentation defects (absent xanthophore and iridophore pigmentation, reduced melanin) and microphthalmia due to failure of cell cycle exit of proliferative retinoblasts. Pigmentation defects arise from GTP pathway deficiency while microphthalmia arises from ATP pathway deficiency, with S phase of retinoblasts prolonged in ATP-deficient conditions. Zebrafish recessive mutation analysis; genetic complementation; purine metabolite rescue experiments Development (Cambridge, England) High 19570845
2008 Active site mutagenesis in CHO cells demonstrated that glutamate-75 is essential for GARS enzymatic activity and glycine-684 is essential for AIRS enzymatic activity of the trifunctional GART protein. Site-directed mutagenesis in CHO-K1 cells; purine auxotrophy selection; mRNA and protein analysis Gene Medium 19007868
2023 GART was found to have a novel methyltransferase activity with enzymatic activity center at residue E948. GART methylates RuvB-like AAA ATPase 1 (RUVBL1) at its K7 site, enhancing RUVBL1 protein stability, which consequently aberrantly activates Wnt/β-catenin signaling to promote tumor stemness in colorectal cancer. In vitro methyltransferase assay; site-directed mutagenesis (E948); Co-IP; western blot; in vivo xenograft and PDX models Advanced science (Weinheim, Baden-Wurttemberg, Germany) Medium 37439412
2025 Under glucose limitation, AMPK phosphorylates GART at Ser440, facilitating GART's interaction with UCK2. GART's catalytic activity generates tetrahydrofolate (THF), which inhibits ILKAP phosphatase activity, thereby preventing ILKAP from removing AKT1-mediated UCK2-Ser254 phosphorylation. Loss of UCK2-Ser254 phosphorylation causes Trim21-mediated UCK2 polyubiquitination and degradation. Thus GART (via both direct binding and THF production) maintains UCK2 stability and pyrimidine salvage synthesis for tumor growth under glucose limitation. Co-IP; western blot; phosphorylation assays; siRNA knockdown; biochemical reconstitution of AMPK phosphorylation; xenograft models Oncogene Medium 39865175
2025 GART binds to and stabilizes HSP90α protein, thereby upregulating its client protein CDK6, and activates the Wnt/β-catenin pathway to promote cell proliferation and stemness gene expression in multiple myeloma cells. Co-IP; lentivirus-based overexpression; siRNA knockdown; in vivo plasmacytoma mouse model; CDX xenograft model European journal of pharmacology Medium 40185325
2025 GART knockdown in lung cancer cells inhibits activation of the PAICS-Akt-β-catenin pathway, suppressing cell proliferation and migration both in vitro and in vivo, placing GART upstream of PAICS in this signaling axis. siRNA knockdown; qRT-PCR; western blot; CCK-8, colony formation, wound healing assays; xenograft tumor model Frontiers in oncology Low 40201340
2016 GART inhibition in intestinal epithelial cells (IECs) induces apoptosis and suppresses IEC migration through activation of the MEKK3-MKK3-p38 MAPK pathway, followed by dysregulation of p53 and PUMA, establishing GART as a pro-survival regulator of intestinal epithelial barrier integrity. siRNA knockdown in cultured IECs; apoptosis assays; migration assays; western blot for pathway components Apoptosis Low 27718035
2023 GART inhibition (via siRNA or lometrexol) induces ERα degradation and prevents breast cancer cell proliferation; VGLL3 transcription factor induces GART expression, and GART knockdown or lometrexol represses proliferation of VGLL3-high cancer cells in a manner rescuable by IMP supplementation. siRNA-based functional screen; metabolomic analysis; VGLL3 stable overexpression; lometrexol inhibitor; IMP rescue experiments Frontiers in endocrinology / Journal of cellular biochemistry Medium 35434822 37143728
2023 Gart in Drosophila is expressed in glia, fat body, and gut, and is directly transcriptionally regulated by the CLOCK/CYCLE heterodimer via canonical E-box elements. CLK in glia, fat body, and gut positively regulates peripheral Gart, while brain-core CLK negatively controls peripheral Gart. Gut Gart maintains endogenous feeding rhythms and food intake, while glia/fat body Gart regulates energy homeostasis. Tissue-specific Gart knockdown; reporter assays for E-box-driven Gart transcription; behavioral feeding assays; genetic epistasis Cell reports Medium 37531254
2026 In Drosophila, NPF signaling via its receptor NPFR positively regulates Gart expression specifically in the intestine, and Gart activity exerts negative feedback on NPF expression, forming a reciprocal regulatory loop. Genetic epistasis experiments demonstrate that gut Gart acts downstream of NPF. Peripheral (fat body/gut-derived) NPF, rather than brain-derived NPF, is the primary systemic signal driving this loop. Genetic epistasis; tissue-specific knockdown; feeding behavior assays; NPF receptor manipulation Insects Medium 42188194
1991 The human GART gene (spanning ~40 kb on chromosome 21q22.1) encodes the trifunctional protein with GARS, GART, and AIRS activities and is functional when transferred into GARS- or GARS/AIRS-deficient CHO cells via YAC DNA, confirming that a single gene complements all three enzymatic deficiencies. YAC DNA transfer by lipofection and spheroplast fusion into mutant CHO cells; complementation of purine auxotrophy; restriction analysis of transferred DNA The EMBO journal High 2050105

Source papers

Stage 0 corpus · 40 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1987 Conserved arrangement of nested genes at the Drosophila Gart locus. Genetics 162 3123310
2013 Detection of haplotypes associated with prenatal death in dairy cattle and identification of deleterious mutations in GART, SHBG and SLC37A2. PloS one 121 23762392
2009 Zebrafish mutations in gart and paics identify crucial roles for de novo purine synthesis in vertebrate pigmentation and ocular development. Development (Cambridge, England) 64 19570845
1991 Cloning and in vivo expression of the human GART gene using yeast artificial chromosomes. The EMBO journal 64 2050105
1997 The human GARS-AIRS-GART gene encodes two proteins which are differentially expressed during human brain development and temporally overexpressed in cerebellum of individuals with Down syndrome. Human molecular genetics 62 9328467
1986 The Saccharomyces cerevisiae ADE5,7 protein is homologous to overlapping Drosophila melanogaster Gart polypeptides. Journal of molecular biology 48 3097325
2008 Plasticity-related genes (PRGs/LRPs): a brain-specific class of lysophospholipid-modifying proteins. Biochimica et biophysica acta 39 18472022
2010 Structural studies of tri-functional human GART. Nucleic acids research 38 20631005
1990 Genetic analysis of the adenosine3 (Gart) region of the second chromosome of Drosophila melanogaster. Genetics 35 2108904
2012 Current views on regulation and function of plasticity-related genes (PRGs/LPPRs) in the brain. Biochimica et biophysica acta 30 23388400
2000 Organization and conservation of the GART/SON/DONSON locus in mouse and human genomes. Genomics 28 10950926
1993 High-resolution mapping of D16led-1, Gart, Gas-4, Cbr, Pcp-4, and Erg on distal mouse chromosome 16. Genomics 28 8406490
2023 GART Functions as a Novel Methyltransferase in the RUVBL1/β-Catenin Signaling Pathway to Promote Tumor Stemness in Colorectal Cancer. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 27 37439412
2010 Correlation between polymorphisms in ADSL and GARS-AIRS-GART genes with inosine 5'-monophosphate (IMP) contents in Beijing-you chickens. British poultry science 21 21058063
1991 Repair of UV-induced pyrimidine dimers in the individual genes Gart, Notch and white from Drosophila melanogaster cell lines. Nucleic acids research 18 1648203
1991 Transfer of the human HPRT and GART genes from yeast to mammalian cells by microinjection of YAC DNA. Somatic cell and molecular genetics 17 1767336
2008 Mutations in the Chinese hamster ovary cell GART gene of de novo purine synthesis. Gene 15 19007868
2023 Regulation of feeding and energy homeostasis by clock-mediated Gart in Drosophila. Cell reports 14 37531254
1997 The repair of UV-induced cyclobutane pyrimidine dimers in the individual genes Gart, Notch and white from isolated brain tissue of Drosophila melanogaster. Mutation research 14 9088344
2009 Combined effect of mutations in ADSL and GARS-AIRS-GART genes on IMP content in chickens. British poultry science 12 19946821
2007 Potential interaction between the GARS-AIRS-GART Gene and CP2/LBP-1c/LSF transcription factor in Down syndrome-related Alzheimer disease. Cellular and molecular neurobiology 12 17902044
2001 Proton transfer dynamics of GART: the pH-dependent catalytic mechanism examined by electrostatic calculations. Protein science : a publication of the Protein Society 12 11604543
2022 VGLL3 increases the dependency of cancer cells on de novo nucleotide synthesis through GART expression. Journal of cellular biochemistry 11 35434822
2014 GART expression in rat spinal cord after injury and its role in inflammation. Brain research 11 24709117
2016 GART mediates the renewal of intestinal epithelial barrier via p38/p53/PUMA cascade in colitis. Apoptosis : an international journal on programmed cell death 10 27718035
2023 A functional genetic screen for metabolic proteins unveils GART and the de novo purine biosynthetic pathway as novel targets for the treatment of luminal A ERα expressing primary and metastatic invasive ductal carcinoma. Frontiers in endocrinology 8 37143728
1988 Mapping of bovine PRGS and PAIS genes in hybrid somatic cells: syntenic conservation with human chromosome 21. Biochemical genetics 8 3377762
1992 Unusual organizational features of the Drosophila Gart locus are not conserved within Diptera. Journal of molecular evolution 7 1518084
2025 The protection of UCK2 protein stability by GART maintains pyrimidine salvage synthesis for HCC growth under glucose limitation. Oncogene 5 39865175
2003 The pH dependence of stability of the activation helix and the catalytic site of GART. Biophysical chemistry 4 14499900
2025 GART promotes the proliferation and migration of human non-small cell lung cancer cell lines A549 and H1299 by targeting PAICS-Akt-β-catenin pathway. Frontiers in oncology 3 40201340
2005 Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1-q12.2. Gene 3 15777723
2009 Phylogenetic analysis and in silico characterization of the GARS-AIRS-GART gene which codes for a tri-functional enzyme protein involved in de novo purine biosynthesis. Molecular biotechnology 2 19301155
1993 The glycinamide ribonucleotide transformylase (GART) gene is not responsible for familial amyotrophic lateral sclerosis. Neuromuscular disorders : NMD 2 8358240
2025 GART promotes multiple myeloma malignancy via tumor stemness mediated by activating the HSP90α/CDK6/β-catenin axis. European journal of pharmacology 1 40185325
2024 Multiple Abdominal Desmoplastic Small Round-Cell Tumors Treated With Fan Beam Computed Tomography-Guided Adaptive Radiotherapy (FBCT-gART): A Case Report. Cureus 1 39308846
2012 No Evidence for Mutations that Deregulate GARS-AIRS-GART Protein Levels in Children with Down Syndrome. Indian journal of clinical biochemistry : IJCB 1 23277712
2026 Licochalcone A from Ma-Xing-Shi-Gan decoction to prevent Asthma through the inhibition of ferroptosis CD4+ T Cell by GART/HSP90α signaling pathway. International immunopharmacology 0 41967212
2026 NPF-Driven Gart Expression Fuels Gut Absorption and Modulates Feeding via a Negative Feedback Loop. Insects 0 42188194
2025 PgFLC/PgFT Regulation by the PgDOF3.4-PgULT1-PgLHP1 Module Mediates the Low Temperature to Promote Flowering in Physalis grisea. Plant, cell & environment 0 41286559

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