関: GAR TFase、phosphoribosylglycinamide formyltransferase

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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2015/01/28 15:19:32」(JST)

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Phosphoribosylglycinamide formyltransferase (EC 2.1.2.2, 2-amino-N-ribosylacetamide 5'-phosphate transformylase, GAR formyltransferase, GAR transformylase, glycinamide ribonucleotide transformylase, GAR TFase, 5,10-methenyltetrahydrofolate:2-amino-N-ribosylacetamide ribonucleotide transformylase) is an enzyme with system name 10-formyltetrahydrofolate:5'-phosphoribosylglycinamide N-formyltransferase.^[1]^[2]^[3] This enzyme catalyses the following chemical reaction

10-formyltetrahydrofolate + N¹-(5-phospho-D-ribosyl)glycinamide tetrahydrofolate + N²-formyl-N¹-(5-phospho-D-ribosyl)glycinamide

Overall reaction of GAR transformylase

This THF dependent enzyme catalyzes a nucleophilic acyl substitution of the formyl group from 10-formyltetrahydrofolate (fTHF) to N¹-(5-phospho-D-ribosyl)glycinamide (GAR) to form N²-formyl-N¹-(5-phospho-D-ribosyl)glycinamide (fGAR) as shown above.^[4] This reaction plays an important role in the formation of purine through the de novo purine biosynthesis pathway. This pathway creates inosine monophosphate (IMP), a precursor to adenosine monophosphate (AMP) and guanosine monophosphate (GMP). AMP is a building block for important energy carriers such as ATP, NAD⁺ and FAD, and signaling molecules such as cAMP. GARTfase's role in de novo purine biosynthesis makes it a target for anti-cancer drugs^[5] and its overexpression during postnatal development has been connected to Down syndrome.^[6] There are two known types of genes encoding GAR transformylase in E.coli: purN and purT, while only purN is found in humans.^[7] Many residues in the active site are conserved across bacterial, yeast, avian and human enzymes.^[8]

Enzyme Structure

Structure of GAR transformylase in rainbow from N(blue)->C(red). Folate binding loop is highlighted in black. GAR substrate (red) and THF analogue (blue) are shown in binding pockets.^[9]

In humans, GARTfase is part of trifunctional enzyme which also includes glycinamide ribnucleotide synthase (GARS) and aminoimidazole ribonucleotide synthetase (AIRS). This protein (110kDa) catalyzes steps 2, 3 and 5 of de novo purine biosynthesis. The proximity of these enzyme units and flexibility of the protein serves to increase pathway throughput. GARTfase is located on the C-terminal end of the protein.^[10]

Human GARTfase has been crystallized by vapor-diffusion sitting drop method and imaged at the Stanford Synchrotron Radiation Laboratory (SSRL) by at least two groups.^[5]^[11]

The structure can be described by two subdomains which are connected by a seven-stranded beta sheet. The N- terminal domain consists of a Rossman type mononucleotide fold, with a four strand part of the beta sheet surrounded on each side by two alpha helices. The beta sheet continues into the C terminal domain, where on one side it is covered by a long alpha helix and on the other it is partially exposed to solvent. It is the cleft between the two subdomains where the active site lies.^[8]

The cleft consists of the GAR binding site and the folate binding pocket. The folate binding pocket is delineated by pteridine-binding cleft, the formyl transfer region and the benzoylglutamate region which bind thepteridine head and a benzoylglutamate tail connected by a formyl bound nitrogen of fTHF. This folate binding region has been the subject of much research because its inhibition by small molecules has led to the discovery of antineoplastic drugs. The folate binding loop has been shown to change conformation depending on the pH of solution and as such Human GAR transformylase shows highest activity around pH 7.5-8. Lower pH (~4.2) conditions change the conformation of the substrate (GAR) binding loops as well.^[11]

Mechanism

Mechanism of purN GARTfase

GAR transformylase water assisted mechanism suggested by Qiao et al

Klein et al first suggested a water molecule assisted mechanism. A single water molecule possibly held in place by hydrogen bonding with the carboxylate group of the persistent Asp144 residue transfers protons from the GAR-N to the THF-N. The nucleophilic nitrogen on the terminal amino group of GAR attacks the carbonyl carbon of the formyl group on THF pushing negative charge onto the oxygen. Klein suggests that His108 stabilizes the transition state by hydrogen bonding with the negatively charged oxygen and that the reformation of the carbonyl double bond results in breaking the THF-N - formyl bond. Calculations by Qiao et al suggest that the water assisted stepwise proton transfer from Gar-N to THF-N is 80-100 kj/mol more favorable than the concerted transfer suggested by Klein. The mechanism shown is suggested by Qiao et al, whom admittedly did not consider surrounding residues in their calculations.^[12]^[13]

Mechanism of purT GARTfase

Studies of the purT variant of GAR transformylase in E.coli found that the reaction proceeds through a formyl phosphate intermediate. While the in vitro reaction can proceed without THF, overall the in vivo reaction is the same.^[14]

Involvement in de novo Purine Biosynthesis

GART catalyzes the third step in the ten step de novo purine biosynthesis, the formation of N²-formyl-N¹-(5-phospho-D-ribosyl)glycinamide (fGAR) by formyl addition to N¹-(5-phospho-D-ribosyl)glycinamide (GAR).^[3] In E. coli, the purN enzyme is a 23 kDa protein^[15] but in humans it is part of a trifunctional protein of 110 kDa which includes AIRS and GARS functionalities.^[10] This protein catalyzes three different steps of the de novo purine pathway.

Disease Relevance

Cancer Target

Due to their increased growth rate and metabolic requirements, cancer cells rely on de novo nucleotide biosynthesis to achieve levels of AMP and GMP necessary.^[16] Being able to block any of the steps of the de novo purine pathway would present significant reduction in tumor growth. Studies have been done both on the substrate binding^[17] and folate binding site^[18] to find inhibitors.

Down Syndrome

GARTfase is suspected to be connected with Down syndrome. The gene encoding the trifunctional protein human GARS-AIRS-GART is located on chromosome 21q22.1, in the Down syndrome critical region. The protein is overexpressed in the cerebellum during the postnatal development of individuals with Down syndrome. Typically, this protein is undetectable in cerebellum shortly after birth, but found in high levels in prenatal development.^[6]^[19]

References

^ Hartman, S. C.; Buchanan, J. M. (1959). "Biosynthesis of the purines. XXVI. The identification of the formyl donors of the transformylation reactions". The Journal of biological chemistry 234 (7): 1812–6. PMID 13672969. edit
^ Smith, G. K.; Benkovic, P. A.; Benkovic, S. J. (1981). "L(-)-10-Formyltetrahydrofolate is the cofactor for glycinamide ribonucleotide transformylase from chicken liver". Biochemistry 20 (14): 4034–6. doi:10.1021/bi00517a013. PMID 7284307. edit
^ ^a ^b Warren, L; Buchanan, J. M. (1957). "Biosynthesis of the purines. XIX. 2-Amino-N-ribosylacetamide 5'-phosphate (glycinamide ribotide) transformylase". The Journal of biological chemistry 229 (2): 613–26. PMID 13502326. edit
^ McMurry, J. and Tadhg, B. The Organic Chemistry of Biological Pathways
^ ^a ^b Connelly, S.; Demartino, J. K.; Boger, D. L.; Wilson, I. A. (2013). "Biological and Structural Evaluation of 10R- and 10S-Methylthio-DDACTHF Reveals a New Role for Sulfur in Inhibition of Glycinamide Ribonucleotide Transformylase". Biochemistry 52 (30): 5133. doi:10.1021/bi4005182. edit
^ ^a ^b Banerjee, D; Nandagopal, K (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 27 (8): 1117–26. doi:10.1007/s10571-007-9217-2. PMID 17902044. edit
^ Nygaard, P; Smith, J. M. (1993). "Evidence for a novel glycinamide ribonucleotide transformylase in Escherichia coli". Journal of bacteriology 175 (11): 3591–7. PMC 204760. PMID 8501063. edit
^ ^a ^b Chen, P; Schulze-Gahmen, U; Stura, E. A.; Inglese, J; Johnson, D. L.; Marolewski, A; Benkovic, S. J.; Wilson, I. A. (1992). "Crystal structure of glycinamide ribonucleotide transformylase from Escherichia coli at 3.0 a resolution. A target enzyme for chemotherapy". Journal of Molecular Biology 227 (1): 283–92. doi:10.1016/0022-2836(92)90698-j. PMID 1522592. edit
^ Zhang, Y., Desharnais, J., Boger, D.L., Wilson, I.A. (2005) "Human GAR Tfase complex structure with 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid and substrate beta-GAR." Unplubished. PDB: 1RBY.
^ ^a ^b Welin, M; Grossmann, J. G.; Flodin, S; Nyman, T; Stenmark, P; Trésaugues, L; Kotenyova, T; Johansson, I; Nordlund, P; Lehtiö, L (2010). "Structural studies of tri-functional human GART". Nucleic Acids Research 38 (20): 7308–19. doi:10.1093/nar/gkq595. PMC 2978367. PMID 20631005. edit
^ ^a ^b Zhang, Y; Desharnais, J; Greasley, S. E.; Beardsley, G. P.; Boger, D. L.; Wilson, I. A. (2002). "Crystal structures of human GAR Tfase at low and high pH and with substrate beta-GAR". Biochemistry 41 (48): 14206–15. doi:10.1021/bi020522m. PMID 12450384. edit
^ Klein, C; Chen, P; Arevalo, J. H.; Stura, E. A.; Marolewski, A; Warren, M. S.; Benkovic, S. J.; Wilson, I. A. (1995). "Towards structure-based drug design: Crystal structure of a multisubstrate adduct complex of glycinamide ribonucleotide transformylase at 1.96 a resolution". Journal of Molecular Biology 249 (1): 153–75. doi:10.1006/jmbi.1995.0286. PMID 7776369. edit
^ Qiao, Q. A.; Jin, Y; Yang, C; Zhang, Z; Wang, M (2005). "A quantum chemical study on the mechanism of glycinamide ribonucleotide transformylase inhibitor: 10-Formyl-5,8,10-trideazafolic acid". Biophysical Chemistry 118 (2-3): 78–83. doi:10.1016/j.bpc.2005.07.001. PMID 16198047. edit
^ Marolewski, A. E.; Mattia, K. M.; Warren, M. S.; Benkovic, S. J. (1997). "Formyl phosphate: A proposed intermediate in the reaction catalyzed by Escherichia coli PurT GAR transformylase". Biochemistry 36 (22): 6709–16. doi:10.1021/bi962961p. PMID 9184151. edit
^ Nixon, A. E.; Benkovic, S. J. (2000). "Improvement in the efficiency of formyl transfer of a GAR transformylase hybrid enzyme". Protein engineering 13 (5): 323–7. doi:10.1093/protein/13.5.323. PMID 10835105. edit
^ Tong, X; Zhao, F; Thompson, C. B. (2009). "The molecular determinants of de novo nucleotide biosynthesis in cancer cells". Current Opinion in Genetics & Development 19 (1): 32–7. doi:10.1016/j.gde.2009.01.002. PMC 2707261. PMID 19201187. edit
^ Antle, V. D.; Donat, N; Hua, M; Liao, P. L.; Vince, R; Carperelli, C. A. (1999). "Substrate specificity of human glycinamide ribonucleotide transformylase". Archives of biochemistry and biophysics 370 (2): 231–5. doi:10.1006/abbi.1999.1428. PMID 10577357. edit
^ Costi, M. P.; Ferrari, S (2001). "Update on antifolate drugs targets". Current drug targets 2 (2): 135–66. doi:10.2174/1389450013348669. PMID 11469716. edit
^ Brodsky, G; Barnes, T; Bleskan, J; Becker, L; Cox, M; Patterson, D (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 6 (12): 2043–50. doi:10.1093/hmg/6.12.2043. PMID 9328467. edit

External links

Phosphoribosylglycinamide formyltransferase at the US National Library of Medicine Medical Subject Headings (MeSH)

English Journal

Biological and structural evaluation of 10R- and 10S-methylthio-DDACTHF reveals a new role for sulfur in inhibition of glycinamide ribonucleotide transformylase.

Connelly S, DeMartino JK, Boger DL, Wilson IA.Author information Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.AbstractGlycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10S- and 10R-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (Ki = 210 nM for 10R, and Ki = 180 nM for 10S) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC₅₀ = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC₅₀ = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diastereoisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. In silico docking simulations of three other sulfur-containing folate analogues also indicate that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase but also the selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for the future design of potent and selective anti-folate drugs that target GAR Tfase.
Biochemistry.Biochemistry.2013 Jul 30;52(30):5133-44. doi: 10.1021/bi4005182. Epub 2013 Jul 19.
Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of bo
PMID 23869564

Reduced folate carrier and folylpolyglutamate synthetase, but not thymidylate synthase predict survival in pemetrexed-treated patients suffering from malignant pleural mesothelioma.

Mairinger F, Vollbrecht C, Halbwedl I, Hatz M, Stacher E, Gülly C, Quehenberger F, Stephan-Falkenau S, Kollmeier J, Roth A, Mairinger T, Popper H.Author information Department of Pathology and Neuropathology, University Hospital Essen, University of Duisberg-Essen, Essen, Germany. fabian.mairinger@uk-essen.deAbstractBACKGROUND: Malignant mesothelioma is a highly aggressive tumor arising from mesothelial-lined surfaces, most often in the pleura cavities. Antifolates belong to the most effective cytotoxic drugs for malignant pleural mesothelioma (MPM) treatment. Pemetrexed is an antifolate inhibiting different folate pathway genes (thymidylate synthase [TS], dihydrofolate reductase, glycinamide ribonucleotide formyltransferase [GARFT], and aminoimidazole carboxamide ribonucleotide formyltransferase, [AICARFT]). Increased activity of pemetrexed occurs by folylpolyglutamate synthetase (FPGS), intracellular transport by reduced folate carrier (RFC). The aim of the study was to explore potential correlations between TS, GARFT, AICARFT, RFC, and FPGS levels in MPM and associations with clinical benefit from pemetrexed treatment.
Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.J Thorac Oncol.2013 May;8(5):644-53. doi: 10.1097/JTO.0b013e318287c224.
BACKGROUND: Malignant mesothelioma is a highly aggressive tumor arising from mesothelial-lined surfaces, most often in the pleura cavities. Antifolates belong to the most effective cytotoxic drugs for malignant pleural mesothelioma (MPM) treatment. Pemetrexed is an antifolate inhibiting different fo
PMID 23449276

Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter.

Cherian C, Kugel Desmoulin S, Wang L, Polin L, White K, Kushner J, Stout M, Hou Z, Gangjee A, Matherly LH.Author information Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201, USA.AbstractPURPOSE: We examined whether the novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate, compound 2, might be an effective treatment for malignant pleural mesothelioma (MPM), reflecting its selective membrane transport by the proton-coupled folate transport (PCFT) over the reduced folate carrier (RFC).
Cancer chemotherapy and pharmacology.Cancer Chemother Pharmacol.2013 Apr;71(4):999-1011. doi: 10.1007/s00280-013-2094-0. Epub 2013 Feb 15.
PURPOSE: We examined whether the novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate, compound 2, might be an effective treatment for malignant pleural mesothelioma (MPM), reflecting its selective membrane transport by the proton-coupled folate transport (PCFT) over the reduced folate c
PMID 23412628

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★リンクテーブル★

リンク元	「グリシンアミドリボヌクレオチドホルミルトランスフェラーゼ」「GAR TFase」「phosphoribosylglycinamide formyltransferase」

「グリシンアミドリボヌクレオチドホルミルトランスフェラーゼ」

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Phosphoribosylglycinamide formyltransferase
Identifiers
EC number	2.1.2.2
CAS number	2604945
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BRENDA	BRENDA entry
ExPASy	NiceZyme view
KEGG	KEGG entry
MetaCyc	metabolic pathway
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