|Gene:||purN||Accession Numbers: EG10799 (EcoCyc), b2500, ECK2496|
Synonyms: ade(c), ade
E. coli contains two different phosphoribosylglycinamide (GAR) transformylases, both of which can catalyze the third step in de novo purine biosynthesis. The transformylase encoded by purN utilizes 10-formyl-tetrahydrofolate as the formyl donor. The second transformylase encoded by purT utilizes formate, which is provided by PurU-catalyzed hydrolysis of 10-formyl-tetrahydrofolate [Nagy93]. The existence of these two transformylase enzymes was determined by mutant studies. A strain containing a knockout insertion in purN did not result in purine auxotrophy [Smith87]. Only mutants defective in both purN and purT required exogenously added purine for growth [Nygaard93]. There is no significant homology between the two transformylases [Marolewski94].
In early work using E. coli B, 10-formyltetrahydrofolate was identified as the formyl donor for this enzyme [Dev78]. Later work using E. coli K-12 showed that the enzyme is monofunctional and catalyzes a reaction involving the transfer of a formyl group from 10-formyl-tetrahydrofolate to the amino nitrogen of 5-phospho-ribosyl-glycineamide (GAR), producing 5'-phosphoribosyl-N-formylglycineamide (FGAR) and tetrahydrofolate [Dev78, Stura89, Inglese90a]. The PurN catalyzed reaction is one of two reactions in the de novo purine biosynthesis pathway that require reduced folate cofactors. The other is the AICAR transformylase activity of the bifunctional product of purH.
A random sequential kinetic mechanism for PurN was proposed [Shim98]. Asp144 was identified as a functional active site residue [Inglese90] and catalytically important residues Asn106, His108, Ser135 and Asp144, were also identified by site-directed mutagenesis [Warren96, Shim99]. The crystal structure of the enzyme has been solved [Stura89, Almassy92, Chen92, Su98, Greasley99]. Although E. coli PurN was shown to be a monomer in solution [Inglese90a], it was a dimer in the crystal structure [Chen92]. The important role of E. coli PurN in purine de novo biosynthesis has made it a model for inhibitor development [Almassy92, Chen92, Greasley99]. The human enzyme is a target for the development of antineoplastic drugs [DeMartino08].
A novel, engineered PurN heterodimer has been produced with activity and kinetics similar to wild-type [Liu00]. A novel, engineered hybrid PurN/PurU enzyme was also constructed and its phosphoribosylglycinamide formyltransferase (GAR transformylase) efficiency was improved by combinatorial mutagenesis [Nixon00].
The subcellular location of E. coli PurN was studied using GFP-labeled protein. It was shown to be diffused throughout the cytoplasm, thus eliminating the possibility of complex formation with other pathway enzymes mediated by cytoskeletal or membrane elements [Gooljarsingh01].
Review: Jensen, K.F., G. Dandanell, B. Hove-Jensen, and M. Willemoes (2008) "Nucleotides, Nucleosides and Nucleobases" EcoSal 3.6.2 [ECOSAL]
|Map Position: [2,620,256 -> 2,620,894] (56.47 centisomes, 203°)||Length: 639 bp / 212 aa|
Molecular Weight of Polypeptide: 23.238 kD (from nucleotide sequence), 25.0 kD (experimental) [Inglese90a ]
Isozyme Sequence Similarity:
phosphoribosylglycinamide formyltransferase 2: NO
Unification Links: ASAP:ABE-0008232 , CGSC:17623 , EchoBASE:EB0792 , EcoGene:EG10799 , EcoliWiki:b2500 , ModBase:P08179 , OU-Microarray:b2500 , PortEco:purN , PR:PRO_000023643 , Pride:P08179 , Protein Model Portal:P08179 , RefSeq:NP_416995 , RegulonDB:EG10799 , SMR:P08179 , String:511145.b2500 , UniProt:P08179
Relationship Links: InterPro:IN-FAMILY:IPR001555 , InterPro:IN-FAMILY:IPR002376 , InterPro:IN-FAMILY:IPR004607 , PDB:Structure:1C2T , PDB:Structure:1C3E , PDB:Structure:1CDD , PDB:Structure:1CDE , PDB:Structure:1GAR , PDB:Structure:1GRC , PDB:Structure:1JKX , PDB:Structure:2GAR , PDB:Structure:3GAR , Pfam:IN-FAMILY:PF00551 , Prosite:IN-FAMILY:PS00373
In Paralogous Gene Group: 397 (3 members)
|Biological Process:||GO:0006974 - cellular response to DNA damage stimulus
GO:0006164 - purine nucleotide biosynthetic process [UniProtGOA11a]
GO:0006189 - 'de novo' IMP biosynthetic process [UniProtGOA12, GOA06, GOA01a]
GO:0009058 - biosynthetic process [GOA01a]
GO:0032259 - methylation [GOA01a]
|Molecular Function:||GO:0004644 - phosphoribosylglycinamide formyltransferase activity
[GOA06, GOA01, GOA01a, Inglese90a]
GO:0008168 - methyltransferase activity [GOA01a]
GO:0016740 - transferase activity [UniProtGOA11a]
GO:0016742 - hydroxymethyl-, formyl- and related transferase activity [GOA01a]
|Cellular Component:||GO:0005829 - cytosol
GO:0005737 - cytoplasm
|MultiFun Terms:||metabolism → biosynthesis of building blocks → nucleotides → purine biosynthesis|
|Growth Medium||Growth?||T (°C)||O2||pH||Osm/L||Growth Observations|
|LB enriched||Yes||37||Aerobic||6.95||Yes [Gerdes03, Comment 1]|
|LB Lennox||Yes||37||Aerobic||7||Yes [Baba06, Comment 2]|
|M9 medium with 1% glycerol||Yes||37||Aerobic||7.2||0.35||Yes [Joyce06, Comment 3]|
|MOPS medium with 0.4% glucose||Yes||37||Aerobic||7.2||0.22||Yes [Baba06, Comment 2] |
Yes [Feist07, Comment 4]
Enzymatic reaction of: phosphoribosylglycinamide formyltransferase
Synonyms: GART, GAR transformylase, 5'-phosphoribosylglycinamide transformylase, GAR TFase, glycinamide ribonucleotide transformylase
EC Number: 126.96.36.199
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.
This reaction is reversible. [Shim98]
The enzyme was assayed using either the natural cofactor, or N10-formyl-5,8-dideazafolate [Inglese90a].
The kcat in the forward direction is substantially higher than in the reverse direction [Shim98].
|Protein-Segment||11 -> 13|
|Protein-Segment||89 -> 92|
|Protein-Segment||140 -> 144|
|Protein-Segment||170 -> 173|
10/20/97 Gene b2500 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10799; confirmed by SwissProt match.
Almassy92: Almassy RJ, Janson CA, Kan CC, Hostomska Z (1992). "Structures of apo and complexed Escherichia coli glycinamide ribonucleotide transformylase." Proc Natl Acad Sci U S A 1992;89(13);6114-8. PMID: 1631098
Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554
Chen92: Chen P, Schulze-Gahmen U, Stura EA, Inglese J, Johnson DL, Marolewski A, Benkovic SJ, Wilson IA (1992). "Crystal structure of glycinamide ribonucleotide transformylase from Escherichia coli at 3.0 A resolution. A target enzyme for chemotherapy." J Mol Biol 227(1);283-92. PMID: 1522592
DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114
Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909
Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938
Gooljarsingh01: Gooljarsingh LT, Ramcharan J, Gilroy S, Benkovic SJ (2001). "Localization of GAR transformylase in Escherichia coli and mammalian cells." Proc Natl Acad Sci U S A 98(12);6565-70. PMID: 11381136
Greasley99: Greasley SE, Yamashita MM, Cai H, Benkovic SJ, Boger DL, Wilson IA (1999). "New insights into inhibitor design from the crystal structure and NMR studies of Escherichia coli GAR transformylase in complex with beta-GAR and 10-formyl-5,8,10-trideazafolic acid." Biochemistry 38(51);16783-93. PMID: 10606510
Inglese89: Inglese J, Blatchly RA, Benkovic SJ (1989). "A multisubstrate adduct inhibitor of a purine biosynthetic enzyme with a picomolar dissociation constant." J Med Chem 32(5);937-40. PMID: 2709379
Inglese90: Inglese J, Smith JM, Benkovic SJ (1990). "Active-site mapping and site-specific mutagenesis of glycinamide ribonucleotide transformylase from Escherichia coli." Biochemistry 1990;29(28);6678-87. PMID: 2204419
Inglese90a: Inglese J, Johnson DL, Shiau A, Smith JM, Benkovic SJ (1990). "Subcloning, characterization, and affinity labeling of Escherichia coli glycinamide ribonucleotide transformylase." Biochemistry 1990;29(6);1436-43. PMID: 2185839
Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394
Marolewski94: Marolewski A, Smith JM, Benkovic SJ (1994). "Cloning and characterization of a new purine biosynthetic enzyme: a non-folate glycinamide ribonucleotide transformylase from E. coli." Biochemistry 1994;33(9);2531-7. PMID: 8117714
Morikis01: Morikis D, Elcock AH, Jennings PA, McCammon JA (2001). "Proton transfer dynamics of GART: the pH-dependent catalytic mechanism examined by electrostatic calculations." Protein Sci 10(11);2379-92. PMID: 11604543
Shim99: Shim JH, Benkovic SJ (1999). "Catalytic mechanism of Escherichia coli glycinamide ribonucleotide transformylase probed by site-directed mutagenesis and pH-dependent studies." Biochemistry 38(31);10024-31. PMID: 10433709
Smith87: Smith JM, Daum HA (1987). "Identification and nucleotide sequence of a gene encoding 5'-phosphoribosylglycinamide transformylase in Escherichia coli K12." J Biol Chem 1987;262(22);10565-9. PMID: 3301838
Stura89: Stura EA, Johnson DL, Inglese J, Smith JM, Benkovic SJ, Wilson IA (1989). "Preliminary crystallographic investigations of glycinamide ribonucleotide transformylase." J Biol Chem 264(16);9703-6. PMID: 2656702
Su98: Su Y, Yamashita MM, Greasley SE, Mullen CA, Shim JH, Jennings PA, Benkovic SJ, Wilson IA (1998). "A pH-dependent stabilization of an active site loop observed from low and high pH crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 A." J Mol Biol 281(3);485-99. PMID: 9698564
Watanabe89: Watanabe W, Sampei G, Aiba A, Mizobuchi K (1989). "Identification and sequence analysis of Escherichia coli purE and purK genes encoding 5'-phosphoribosyl-5-amino-4-imidazole carboxylase for de novo purine biosynthesis." J Bacteriol 1989;171(1);198-204. PMID: 2644189
He90: He B, Shiau A, Choi KY, Zalkin H, Smith JM (1990). "Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction." J Bacteriol 1990;172(8);4555-62. PMID: 2198266
Liu11: Liu IF, Aedo S, Tse-Dinh YC (2011). "Resistance to topoisomerase cleavage complex induced lethality in Escherichia coli via titration of transcription regulators PurR and FNR." BMC Microbiol 11;261. PMID: 22152010
Meng90: Meng LM, Kilstrup M, Nygaard P (1990). "Autoregulation of PurR repressor synthesis and involvement of purR in the regulation of purB, purC, purL, purMN and guaBA expression in Escherichia coli." Eur J Biochem 1990;187(2);373-9. PMID: 2404765
Salmon03: Salmon K, Hung SP, Mekjian K, Baldi P, Hatfield GW, Gunsalus RP (2003). "Global gene expression profiling in Escherichia coli K12. The effects of oxygen availability and FNR." J Biol Chem 278(32);29837-55. PMID: 12754220
Smith86a: Smith JM, Daum HA (1986). "Nucleotide sequence of the purM gene encoding 5'-phosphoribosyl-5-aminoimidazole synthetase of Escherichia coli K12." J Biol Chem 1986;261(23);10632-6. PMID: 3015935
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