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Escherichia coli K-12 substr. MG1655 Enzyme: bifunctional folylpolyglutamate synthetase / dihydrofolate synthetase



Gene: folC Accession Numbers: EG10327 (EcoCyc), b2315, ECK2309

Synonyms: dedC

Regulation Summary Diagram: ?

Summary:
During de novo tetrahydrofolate biosynthesis, dihydrofolate synthetase encoded by folC catalyzes the addition of the glutamyl residue to dihydropteroate (7,8-dihydropteroate) to form dihydrofolate (7,8-dihydrofolate monoglutamate) in an ATP-dependent reaction [Griffin64] (see pathway tetrahydrofolate biosynthesis). In E. coli FolC is a bifunctional enzyme that also catalyzes the subsequent additions of L-glutamate to tetrahydrofolate (folylpoly-γ-glutamate synthetase activity). This latter activity can also utilize the 10-formyl and methylene derivatives of tetrahydrofolate as substrate [Bognar85] (see pathway folate polyglutamylation).

The folC gene has been shown to be essential in E. coli because this organism lacks specific folate transporters [Pyne92]. Recombinant FolC has been overexpressed as a soluble protein, purified and characterized [Bognar85].

The enzyme is a member of the Mur synthetase superfamily. The crystal structure of FolC has been determined at 2.1 Å resolution and analysis of crystallographic data suggested the existence of different binding sites for dihydropteridine and tetrahydrofolate on FolC. Its dihydrofolate binding site was found to be distinct from that of Lactobacillus casei, which is a model for the human enzyme, making the E. coli enzyme a possible drug target [Mathieu05].

However, mutant studies have shown that dihydrofolate synthase and folylpolyglutamate synthetase activities could not be separated [Kimlova91, Keshavjee91]. A later mutant study showed that the amino terminal domain of FolC (residues 1-287) binds tetrahydrofolate and dihydroptroate with the same affinity as the intact enzyme [Sheng08].

The antifolate drug trimethoprim is an inhibitor of dihydrofolate reductase (DHFR) encoded by folA. Trimethoprim also indirectly blocks the folylpolyglutamate synthetase activity of FolC due to the accumulation of inhibitory dihydrofolate species that results from DHFR inhibition [Kwon08].

Reviews: Green, J.M. and R.G. Matthews (2007) "Folate Biosynthesis, Reduction, and Polyglutamylation and the Interconversion of Folate Derivatives" EcoSal 3.6.3.6 [ECOSAL]; and reviewed from a structural perspective in [Bermingham02].

Gene Citations: [Bognar87]

Locations: cytosol

Map Position: [2,429,696 <- 2,430,964] (52.37 centisomes)
Length: 1269 bp / 422 aa

Molecular Weight of Polypeptide: 45.406 kD (from nucleotide sequence), 47.0 kD (experimental) [Bognar85 ]

pI: 5.8

Unification Links: ASAP:ABE-0007645 , CGSC:17704 , DIP:DIP-9674N , EchoBASE:EB0323 , EcoGene:EG10327 , EcoliWiki:b2315 , Entrez-gene:945451 , Entrez-Nucleotide:M32445 , Mint:MINT-1301447 , ModBase:P08192 , OU-Microarray:b2315 , PortEco:folC , PR:PRO_000022678 , Pride:P08192 , Protein Model Portal:P08192 , RefSeq:NP_416818.1 , RegulonDB:EG10327 , SMR:P08192 , String:511145.b2315 , UniProt:P08192

Relationship Links: InterPro:IN-FAMILY:IPR001645 , InterPro:IN-FAMILY:IPR004101 , InterPro:IN-FAMILY:IPR013221 , InterPro:IN-FAMILY:IPR018109 , Panther:IN-FAMILY:PTHR11136 , PDB:Structure:1W78 , PDB:Structure:1W7K , Pfam:IN-FAMILY:PF02875 , Pfam:IN-FAMILY:PF08245 , Prosite:IN-FAMILY:PS01011 , Prosite:IN-FAMILY:PS01012

In Paralogous Gene Group: 407 (2 members)

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006761 - dihydrofolate biosynthetic process Inferred from experiment [Keshavjee91]
GO:0009257 - 10-formyltetrahydrofolate biosynthetic process Inferred from experiment [Keshavjee91]
GO:0046656 - folic acid biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA11a, Keshavjee91]
GO:0046901 - tetrahydrofolylpolyglutamate biosynthetic process Inferred from experiment [Keshavjee91]
GO:0006730 - one-carbon metabolic process Inferred by computational analysis [UniProtGOA11a]
GO:0008152 - metabolic process Inferred by computational analysis [UniProtGOA11a]
GO:0009058 - biosynthetic process Inferred by computational analysis [GOA01a]
GO:0009396 - folic acid-containing compound biosynthetic process Inferred by computational analysis [GOA01a, GOA00]
GO:0046654 - tetrahydrofolate biosynthetic process Inferred by computational analysis [UniProtGOA12]
Molecular Function: GO:0004326 - tetrahydrofolylpolyglutamate synthase activity Inferred from experiment Inferred by computational analysis [GOA01, GOA01a, Bognar85]
GO:0008841 - dihydrofolate synthase activity Inferred from experiment Inferred by computational analysis [GOA01, Bognar85]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11a]
GO:0003824 - catalytic activity Inferred by computational analysis [UniProtGOA11a]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
Cellular Component: GO:0005737 - cytoplasm Inferred from experiment [Bognar85]
GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: metabolism biosynthesis of building blocks cofactors, small molecule carriers folic acid
metabolism central intermediary metabolism formyl-THF biosynthesis

Essentiality data for folC knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox No 37 Aerobic 7   No [Baba06, Comment 1]

Credits:
Last-Curated ? 22-May-2012 by Fulcher C , SRI International


Enzymatic reaction of: methylene-tetrahydrofolylpolyglutamate synthetase (bifunctional folylpolyglutamate synthetase / dihydrofolate synthetase)

Synonyms: folylpoly-γ-glutamate synthetase

EC Number: 6.3.2.17

methylene-tetrahydropteroyl-[γ-Glu](n) + L-glutamate + ATP <=> methylene-tetrahydropteroyl-[γ-Glu](n+1) + ADP + phosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

In Pathways: folate polyglutamylation

Summary:
The methylene-THFglutamate synthetase activity is an alternative substrate reaction of folylpolyglutamate synthetase.


Enzymatic reaction of: 10-formyl-tetrahydrofolylpolyglutamate synthetase (bifunctional folylpolyglutamate synthetase / dihydrofolate synthetase)

Synonyms: folylpoly-γ-glutamate synthetase

EC Number: 6.3.2.17

10-formyl-tetrahydropteroyl-[γ-Glu](n) + L-glutamate + ATP <=> 10-formyl-tetrahydropteroyl-[γ-Glu](n+1) + ADP + phosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

In Pathways: folate polyglutamylation

Summary:
The formyl-THFglutamate synthetase activity is an alternative substrate reaction of folylpolyglutamate synthetase.


Enzymatic reaction of: folylpoly-γ-glutamate synthetase (bifunctional folylpolyglutamate synthetase / dihydrofolate synthetase)

Synonyms: tetrahydrofolylpolyglutamate synthetase, FPGS, tetrahydrofolate:L-glutamate γ-ligase (ADP-forming), folylpolyglutamate synthetase

EC Number: 6.3.2.17

tetrahydropteroyl-[γ-Glu](n) + L-glutamate + ATP <=> tetrahydropteroyl-[γ-Glu](n+1) + ADP + phosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown. [Griffin64]

Alternative Substrates for ATP: GTP [Masurekar75 ] , dATP [Masurekar75 ]

In Pathways: folate polyglutamylation

Summary:
ATP, Mg2+ and a monovalent cation are required for product formation. The monovalent cation requirement can be satisfied by K+, NH4+ or Rb+. NH4+ is more effective than K+ at low concentrations, but it becomes inhibitory at higher concentrations. Mn2+ can partially replace Mg2+. GTP and dATP were 20% and 54% as effective as ATP, respectively [Masurekar75].

10-formyl-tetrahydrofolate apeared to be a more efficient substrate than tetrahydrofolate, or methylene-tetrahydrofolate in assays using partially purified enzyme preparations [Masurekar75].

The enzyme was shown to add two glutamyl residues to tetrahydrofolate (tetrahydropteroylglutamate), producing in sequence tetrahydropteroyldiglutamate and tetrahydropteroyltriglutamate [Griffin64].

Cofactors or Prosthetic Groups [Comment 2]: Mg2+ [Masurekar75], K+ [Masurekar75]

Alternative Cofactors for K+: ammonium , Rb+

Alternative Cofactors for Mg2+: Mn2+

Inhibitors (Unknown Mechanism): 10-formyl-tetrahydropterote [Masurekar75] , 7,8-dihydrofolate monoglutamate [Masurekar75] , 10-methyl-5,6,7,8-tetrahydropteroylglutamate [Masurekar75] , N5-methyl-tetrahydropteroyl mono-L-glutamate [Masurekar75] , 7,8-dihydropteroate [Bognar85, Comment 3]


Enzymatic reaction of: dihydrofolate synthetase

Synonyms: dihydropteroate:L-glutamate ligase (ADP-forming)

EC Number: 6.3.2.12

L-glutamate + 7,8-dihydropteroate + ATP <=> ADP + 7,8-dihydrofolate monoglutamate + phosphate + 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.

The reaction is physiologically favored in the direction shown. [Griffin64]

In Pathways: superpathway of chorismate metabolism , superpathway of tetrahydrofolate biosynthesis , tetrahydrofolate biosynthesis

Inhibitors (Competitive): dihydroisopteroate [Ho80] , dihydro-10-thiopteroate [Ho80] , dihydrohomopteroate [Ho80] , 7,8-dihydrofolate monoglutamate [Ho80]

Kinetic Parameters:

Substrate
Km (μM)
Citations
L-glutamate
3900.0
[Bognar85]
7,8-dihydropteroate
0.6
[Bognar85]
ATP
6.9
[Bognar85]


Sequence Features

Feature Class Location Citations Comment
Nucleotide-Phosphate-Binding-Region 56 -> 62
[UniProt10b]
UniProt: ATP; Non-Experimental Qualifier: by similarity;
Sequence-Conflict 326
[Bognar87, UniProt10]
Alternate sequence: V → M; UniProt: (in Ref. 1; AAA23808/AAA23802);
Sequence-Conflict 328
[Nonet87, UniProt10]
Alternate sequence: A → AA; UniProt: (in Ref. 2; AAA23966);
Disulfide-Bond-Site 401, 354
[UniProt10b]
UniProt: Non-Experimental Qualifier: probable;
Sequence-Conflict 391 -> 392
[Nonet87, UniProt10]
Alternate sequence: DA → EP; UniProt: (in Ref. 2; AAA23966);


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b2315 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10327; confirmed by SwissProt match.


References

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

Bermingham02: Bermingham A, Derrick JP (2002). "The folic acid biosynthesis pathway in bacteria: evaluation of potential for antibacterial drug discovery." Bioessays 24(7);637-48. PMID: 12111724

Bognar85: Bognar AL, Osborne C, Shane B, Singer SC, Ferone R (1985). "Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product." J Biol Chem 1985;260(9);5625-30. PMID: 2985605

Bognar87: Bognar AL, Osborne C, Shane B (1987). "Primary structure of the Escherichia coli folC gene and its folylpolyglutamate synthetase-dihydrofolate synthetase product and regulation of expression by an upstream gene." J Biol Chem 262(25);12337-43. PMID: 3040739

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

GOA00: GOA (2000). "Gene Ontology annotation based on Swiss-Prot keyword mapping."

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."

Griffin64: Griffin MJ, Brown GM (1964). "The biosynthesis of folic acid. III. Enzymatic formation of dihydrofolic acid from dihydropteroic acid and of tetrahydropteroylpolyglutamic acid compounds from tetrahydrofolic acid." J Biol Chem 239;310-6. PMID: 14114858

Ho80: Ho RI (1980). "A simple radioassay for dihydrofolate synthetase activity in Escherichia coli and its application to an inhibition study of new pteroate analogs." Methods Enzymol 1980;66;576-81. PMID: 6768963

Keshavjee91: Keshavjee K, Pyne C, Bognar AL (1991). "Characterization of a mutation affecting the function of Escherichia coli folylpolyglutamate synthetase-dihydrofolate synthetase and further mutations produced in vitro at the same locus." J Biol Chem 1991;266(30);19925-9. PMID: 1939056

Kimlova91: Kimlova LJ, Pyne C, Keshavjee K, Huy J, Beebakhee G, Bognar AL (1991). "Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli." Arch Biochem Biophys 284(1);9-16. PMID: 1989505

Kwon08: Kwon YK, Lu W, Melamud E, Khanam N, Bognar A, Rabinowitz JD (2008). "A domino effect in antifolate drug action in Escherichia coli." Nat Chem Biol 4(10);602-8. PMID: 18724364

Masurekar75: Masurekar M, Brown GM (1975). "Partial purification and properties of an enzyme from Escherichia coli that catalyzes the conversion of glutamic acid and 10-formyltetrahydropteroylglutamic acid to 10-formyltetrahydropterol-gamma-glutamyglutamic acid." Biochemistry 1975;14(11);2424-30. PMID: 1095054

Mathieu05: Mathieu M, Debousker G, Vincent S, Viviani F, Bamas-Jacques N, Mikol V (2005). "Escherichia coli FolC structure reveals an unexpected dihydrofolate binding site providing an attractive target for anti-microbial therapy." J Biol Chem 280(19);18916-22. PMID: 15705579

Nonet87: Nonet ML, Marvel CC, Tolan DR (1987). "The hisT-purF region of the Escherichia coli K-12 chromosome. Identification of additional genes of the hisT and purF operons." J Biol Chem 262(25);12209-17. PMID: 3040734

Pyne92: Pyne C, Bognar AL (1992). "Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro." J Bacteriol 174(6);1750-9. PMID: 1548226

Sheng08: Sheng Y, Khanam N, Tsaksis Y, Shi XM, Lu QS, Bognar AL (2008). "Mutagenesis of folylpolyglutamate synthetase indicates that dihydropteroate and tetrahydrofolate bind to the same site." Biochemistry 47(8);2388-96. PMID: 18232714

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

UniProt10b: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 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."


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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 18.5 on Mon Dec 22, 2014, BIOCYC13A.