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MetaCyc Enzyme: phosphoribosylglycinamide formyltransferase 1

Gene: purN Accession Numbers: EG10799 (MetaCyc), b2500, ECK2496

Synonyms: ade(c), ade

Species: Escherichia coli K-12 substr. MG1655

Summary:
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, Inglese90]. 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 [Inglese90a] 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, Chen92a, Su98, Greasley99]. Although E. coli PurN was shown to be a monomer in solution [Inglese90], it was a dimer in the crystal structure [Chen92a]. The important role of E. coli PurN in purine de novo biosynthesis has made it a model for inhibitor development [Almassy92, Chen92a, 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]

Citations: [Morikis01]

Gene Citations: [Watanabe89, Andersen92]

Locations: cytosol

Map Position: [2,620,256 -> 2,620,894]

Molecular Weight of Polypeptide: 23.238 kD (from nucleotide sequence), 25.0 kD (experimental) [Inglese90 ]

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

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006974 - cellular response to DNA damage stimulus Inferred from experiment [Khil02]
GO:0006164 - purine nucleotide biosynthetic process Inferred by computational analysis [UniProtGOA11a]
GO:0006189 - 'de novo' IMP biosynthetic process Inferred by computational analysis [UniProtGOA12, GOA01a]
GO:0009058 - biosynthetic process Inferred by computational analysis [GOA01a]
GO:0032259 - methylation Inferred by computational analysis [GOA01a]
Molecular Function: GO:0004644 - phosphoribosylglycinamide formyltransferase activity Inferred from experiment Inferred by computational analysis [GOA01, GOA01a, Inglese90]
GO:0008168 - methyltransferase activity Inferred by computational analysis [GOA01a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016742 - hydroxymethyl-, formyl- and related transferase activity Inferred by computational analysis [GOA01a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm

MultiFun Terms: metabolism biosynthesis of building blocks nucleotides purine biosynthesis

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International


Enzymatic reaction of: phosphoribosylglycinamide formyltransferase

Synonyms: GART, GAR transformylase, 5'-phosphoribosylglycinamide transformylase, GAR TFase, glycinamide ribonucleotide transformylase

EC Number: 2.1.2.2

an N10-formyl-tetrahydrofolate + N1-(5-phospho-β-D-ribosyl)glycinamide <=> a tetrahydrofolate + N2-formyl-N1-(5-phospho-β-D-ribosyl)glycinamide + H+

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]

In Pathways: superpathway of 5-aminoimidazole ribonucleotide biosynthesis , superpathway of tetrahydrofolate biosynthesis and salvage , 5-aminoimidazole ribonucleotide biosynthesis I , tetrahydrofolate salvage from 5,10-methenyltetrahydrofolate

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Summary:
The enzyme was assayed using either the natural cofactor, or N10-formyl-5,8-dideazafolate [Inglese90].

The kcat in the forward direction is substantially higher than in the reverse direction [Shim98].

Inhibitors (Unknown Mechanism): N10-(bromoacetyl)-5,8-dideazafolate [Inglese90] , β-thiogardideazafolate [Inglese89] , N10-formyl-2,4-diaminodideazafolate [Inglese90]

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
N1-(5-phospho-β-D-ribosyl)glycinamide
12.2
[Inglese90]
N1-(5-phospho-β-D-ribosyl)glycinamide
133.0
[Shim99]
an N10-formyl-tetrahydrofolate
84.8
[Inglese90]

pH(opt): 8 [BRENDA14, Marolewski94], 8.5 [Inglese90]


Sequence Features

Feature Class Location Citations Comment
Protein-Segment 11 -> 13
[UniProt10a]
UniProt: 5'-phosphoribosylglycinamide binding; Sequence Annotation Type: region of interest;
Amino-Acid-Sites-That-Bind 64
[UniProt10]
UniProt: 10-formyltetrahydrofolate;
Mutagenesis-Variant 70
[UniProt10]
Alternate sequence: A; UniProt: Loss of homodimerization. No effect on activity;
Protein-Segment 89 -> 92
[UniProt10a]
UniProt: 10-formyltetrahydrofolate binding; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 106
[UniProt10]
Alternate sequence: Y; UniProt: Loss of activity;
Alternate sequence: L; UniProt: Loss of activity;
Alternate sequence: K; UniProt: Loss of activity;
Alternate sequence: I; UniProt: Loss of activity;
Alternate sequence: H; UniProt: Loss of activity;
Alternate sequence: E; UniProt: Loss of activity;
Alternate sequence: S; UniProt: Reduces activity about 2000- fold;
Alternate sequence: G; UniProt: Reduces activity about 2000- fold;
Alternate sequence: D; UniProt: Reduces activity about 2000- fold;
Alternate sequence: A; UniProt: Reduces activity about 2000- fold;
Amino-Acid-Sites-That-Bind 106
[UniProt10]
UniProt: 10-formyltetrahydrofolate;
Active-Site 108
[Shim99, Inglese90a, UniProt11]
UniProt: Proton donor.
Mutagenesis-Variant 108
[UniProt10]
Alternate sequence: T; UniProt: Reduces activity about 1000- fold;
Alternate sequence: S; UniProt: Reduces activity about 1000- fold;
Alternate sequence: E; UniProt: Reduces activity about 1000- fold;
Alternate sequence: R; UniProt: Loss of activity;
Alternate sequence: Q; UniProt: Loss of activity;
Alternate sequence: N; UniProt: Loss of activity;
Alternate sequence: M; UniProt: Loss of activity;
Alternate sequence: G; UniProt: Loss of activity;
Alternate sequence: A; UniProt: Loss of activity;
Mutagenesis-Variant 119
[UniProt10]
Alternate sequence: A; UniProt: No effect;
Mutagenesis-Variant 121
[UniProt10]
Alternate sequence: Q; UniProt: Increases Km for 5'- phosphoribosylglycinamide 4-fold;
Mutagenesis-Variant 135
[UniProt10]
Alternate sequence: L; UniProt: Reduces activity about 1000-fold;
Alternate sequence: A; UniProt: Reduces activity about 1000-fold;
Mutagenesis-Variant 137
[UniProt10]
Alternate sequence: Q; UniProt: No effect;
Alternate sequence: F; UniProt: No effect;
Protein-Segment 140 -> 144
[UniProt10a]
UniProt: 10-formyltetrahydrofolate binding; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 144
[UniProt10]
Alternate sequence: V; UniProt: Loss of activity;
Alternate sequence: T; UniProt: Loss of activity;
Alternate sequence: R; UniProt: Loss of activity;
Alternate sequence: Q; UniProt: Loss of activity;
Alternate sequence: P; UniProt: Loss of activity;
Alternate sequence: N; UniProt: Loss of activity;
Alternate sequence: L; UniProt: Loss of activity;
Alternate sequence: K; UniProt: Loss of activity;
Alternate sequence: H; UniProt: Loss of activity;
Alternate sequence: F; UniProt: Loss of activity;
Alternate sequence: C; UniProt: Loss of activity;
Alternate sequence: Y; UniProt: Reduces activity about 1000- fold;
Alternate sequence: S; UniProt: Reduces activity about 1000- fold;
Alternate sequence: E; UniProt: Reduces activity about 1000- fold;
Alternate sequence: A; UniProt: Reduces activity about 1000- fold;
Amino-Acid-Site 144
[UniProt10]
UniProt: Raises pKa of active site His; Sequence Annotation Type: site;
Protein-Segment 170 -> 173
[UniProt10a]
UniProt: 5'-phosphoribosylglycinamide binding; Sequence Annotation Type: region of interest;

History:
10/20/97 Gene b2500 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10799; confirmed by SwissProt match.


References

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

Andersen92: Andersen PS, Smith JM, Mygind B (1992). "Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12." Eur J Biochem 1992;204(1);51-6. PMID: 1371255

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014." http://www.brenda-enzymes.org.

Chen92a: 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

DeMartino08: DeMartino JK, Hwang I, Connelly S, Wilson IA, Boger DL (2008). "Asymmetric synthesis of inhibitors of glycinamide ribonucleotide transformylase." J Med Chem 51(17);5441-8. PMID: 18686942

Dev78: Dev IK, Harvey RJ (1978). "N10-Formyltetrahydrofolate is the formyl donor for glycinamide ribotide transformylase in Escherichia coli." J Biol Chem 253(12);4242-4. PMID: 350869

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

ECOSAL: EcoSal "Escherichia coli and Salmonella: Cellular and Molecular Biology." Online edition.

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

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, 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

Inglese90a: 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

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Khil02: Khil PP, Camerini-Otero RD (2002). "Over 1000 genes are involved in the DNA damage response of Escherichia coli." Mol Microbiol 44(1);89-105. PMID: 11967071

Liu00: Liu C, Shim JH, Benkovic SJ (2000). "The Unexpected Catalytic Properties of a Heterodimer of GAR Transformylase." Bioorg Chem 28(6);316-23. PMID: 11352469

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

Nagy93: Nagy PL, McCorkle GM, Zalkin H (1993). "purU, a source of formate for purT-dependent phosphoribosyl-N-formylglycinamide synthesis." J Bacteriol 175(21);7066-73. PMID: 8226647

Nixon00: Nixon AE, Benkovic SJ (2000). "Improvement in the efficiency of formyl transfer of a GAR transformylase hybrid enzyme." Protein Eng 13(5);323-7. PMID: 10835105

Nygaard93: Nygaard P, Smith JM (1993). "Evidence for a novel glycinamide ribonucleotide transformylase in Escherichia coli." J Bacteriol 1993;175(11);3591-7. PMID: 8501063

Shim98: Shim JH, Benkovic SJ (1998). "Evaluation of the kinetic mechanism of Escherichia coli glycinamide ribonucleotide transformylase." Biochemistry 37(24);8776-82. PMID: 9628739

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

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Warren96: Warren MS, Marolewski AE, Benkovic SJ (1996). "A rapid screen of active site mutants in glycinamide ribonucleotide transformylase." Biochemistry 35(27);8855-62. PMID: 8688421

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


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 18.5 on Fri Nov 28, 2014, BIOCYC13B.