Escherichia coli K-12 substr. MG1655 Pathway: folate polyglutamylation
Inferred from experiment

Pathway diagram: folate polyglutamylation

If an enzyme name is shown in bold, there is experimental evidence for this enzymatic activity.

Locations of Mapped Genes:

Schematic showing all replicons, marked with selected genes

Genetic Regulation Schematic

Genetic regulation schematic for folate polyglutamylation

Superclasses: BiosynthesisCofactors, Prosthetic Groups, Electron Carriers BiosynthesisVitamins BiosynthesisFolate Biosynthesis

Folates are required in a variety of reactions (known as one-carbon metabolism) in both bacterial and mammalian tissues, where they act as carriers of one-carbon units in various oxidation states. These one-carbon units are utilized in the biosynthesis of various cellular components, including glycine, methionine, formylmethionine, thymidylate, pantothenate and purine nucleotides.

During folate biosynthesis (as described in superpathway of tetrahydrofolate biosynthesis and salvage) the enzyme dihydrofolate synthetase (encoded in E. coli by folC) adds a glutamate residue to 7,8-dihydropteroate, resulting in 7,8-dihydrofolate, also known as H2PteGlu1. This molecule in turn is reduced by dihydrofolate reductase (FolA) to tetrahydrofolate (H4PteGlu1, or THF). THF can then be converted to several other folate molecules [Sun01]. However, most folate molecules are further modified in cells by successive additions of glutamate residues, forming folate polyglutamtes (or folylpoly-γ-glutamates). Most of the glutamates are added by γ-carboxy-linked polyglutamylation via an amide linkage to the γ-carboxylate group of the folate or folate derivative. Since these isopeptide bonds are not the normal amide bonds they are not hydrolyzed by peptidases or proteases that are specific for α-carboxyl-linked peptide bonds.

The addition of glutamyl residues probably occurs after the reduction of newly synthesized dihydrofolate to tetrahydrofolate and its conversion to other tetrahydrofolate derivatives.

Apparently, the glutamylation of folate residues serves several goals: it prevents the efflux of folates out of the cell, it increases the binding of folate cofactors to the enzymes of folate interconversion and biosynthesis, and in mammals, it allows the accumulation of folates in the mitochondria, which is required for glycine synthesis [Moran99]. Folylpolyglutamates are generally better substrates for folate-dependent enzymes than their monoglutamyl counterparts. Km values decrease with increasing oligo-γ-glutamyl chain length [Shane89]. At least in one case, the vitamin B12-independent methionine synthetase, there is an absolute requirement for the polyglutamate cofactor [Bognar85].

In addition, many folate enzymes are multifunctional and channel one-carbon units between reactions without achieving equilibrium with the cell medium. Therefore, the conjugated oligo-γ-glutamyl chain can potentially regulate the reaction rates, and allows channeling of the substrate between enzymes in a way which controls biosynthetic pathways [Shane89].

Folylpoly-γ-glutamate synthetase (FPGS), the enzyme that catalyzes the conversion of folates to polyglutamates, has been purified from several organisms, including E. coli [Bognar85]. It is a MgATP-dependent enzyme present in all cells. FPGS forms a complex with MgATP, a folate derivative, and glutamate, in an ordered manner whereby the three substrates are added sequentially [Sun01]. In E. coli, FPGS is a bi-functional enzyme, which also catalyzes the addition of glutamate to 7,8-dihydropteroate, generating 7,8,-dihydrofolate (dihydrofolate synthetase, (E.C#

In exponentially growing cells of E. coli folylpoly-γ- glutamates have short glutamate chain lengths: mono- and triglutamate derivatives are most abundant, with tetra-, penta- and hexaglutamate derivatives also present (in order of decreasing abundance). However, in stationary phase, cells contain longer-chain-length folypolyglutamates, with the predominant chain length containing six or seven glutamyl residues. These longer chains are generated by a second enzyme, which adds glutamate moieties in α-linkage to tetrahydropteroyl- triglutamates [Ferone86]. However, this enzyme has not been purified, nor has the gene encoding it been identified.

Both folylpolyglutamate synthetases can accept several different folate derivatives as substrates. It seems that the preferred substrate for the addition of a second glutamate residue is 10-formyl-THF (10-formyl-H4PteGlu1), while the preferred substrate for the addition of a third glutamate residue is the glutamated form of 5,10-methylene-THF (5,10-methylene-H4PteGlu2).

Variants: 4-aminobenzoate biosynthesis, N10-formyl-tetrahydrofolate biosynthesis, superpathway of tetrahydrofolate biosynthesis, tetrahydrofolate biosynthesis

Created 02-Nov-2004 by Caspi R, SRI International


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

Ferone86: Ferone R, Singer SC, Hunt DF (1986). "In vitro synthesis of alpha-carboxyl-linked folylpolyglutamates by an enzyme preparation from Escherichia coli." J Biol Chem 261(35);16363-71. PMID: 3536926

Moran99: Moran RG (1999). "Roles of folylpoly-gamma-glutamate synthetase in therapeutics with tetrahydrofolate antimetabolites: an overview." Semin Oncol 26(2 Suppl 6);24-32. PMID: 10598551

Shane89: Shane B (1989). "Folylpolyglutamate synthesis and role in the regulation of one-carbon metabolism." Vitam Horm 45;263-335. PMID: 2688305

Sun01: Sun X, Cross JA, Bognar AL, Baker EN, Smith CA (2001). "Folate-binding triggers the activation of folylpolyglutamate synthetase." J Mol Biol 310(5);1067-78. PMID: 11501996

Other References Related to Enzymes, Genes, Subpathways, and Substrates of this Pathway

Angelaccio92: Angelaccio S, Pascarella S, Fattori E, Bossa F, Strong W, Schirch V (1992). "Serine hydroxymethyltransferase: origin of substrate specificity." Biochemistry 31(1);155-62. PMID: 1731867

Bairoch93: Bairoch A, Boeckmann B (1993). "The SWISS-PROT protein sequence data bank, recent developments." Nucleic Acids Res. 21:3093-3096. PMID: 8332529

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

Blank14: Blank D, Wolf L, Ackermann M, Silander OK (2014). "The predictability of molecular evolution during functional innovation." Proc Natl Acad Sci U S A 111(8);3044-9. PMID: 24516157

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

Cai95: Cai K, Schirch D, Schirch V (1995). "The affinity of pyridoxal 5'-phosphate for folding intermediates of Escherichia coli serine hydroxymethyltransferase." J Biol Chem 270(33);19294-9. PMID: 7642604

Contestabile00: Contestabile R, Angelaccio S, Bossa F, Wright HT, Scarsdale N, Kazanina G, Schirch V (2000). "Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase." Biochemistry 39(25);7492-500. PMID: 10858298

Delle94: Delle Fratte S, Iurescia S, Angelaccio S, Bossa F, Schirch V (1994). "The function of arginine 363 as the substrate carboxyl-binding site in Escherichia coli serine hydroxymethyltransferase." Eur J Biochem 225(1);395-401. PMID: 7925461

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.

Ferone86a: Ferone R, Hanlon MH, Singer SC, Hunt DF (1986). "alpha-Carboxyl-linked glutamates in the folylpolyglutamates of Escherichia coli." J Biol Chem 261(35);16356-62. PMID: 3536925

Fitzpatrick98: Fitzpatrick TB, Malthouse JP (1998). "A substrate-induced change in the stereospecificity of the serine-hydroxymethyltransferase-catalysed exchange of the alpha-protons of amino acids--evidence for a second catalytic site." Eur J Biochem 252(1);113-7. PMID: 9523719

Florio09: Florio R, Chiaraluce R, Consalvi V, Paiardini A, Catacchio B, Bossa F, Contestabile R (2009). "The role of evolutionarily conserved hydrophobic contacts in the quaternary structure stability of Escherichia coli serine hydroxymethyltransferase." FEBS J 276(1);132-43. PMID: 19019081

Florio09a: Florio R, Chiaraluce R, Consalvi V, Paiardini A, Catacchio B, Bossa F, Contestabile R (2009). "Structural stability of the cofactor binding site in Escherichia coli serine hydroxymethyltransferase--the role of evolutionarily conserved hydrophobic contacts." FEBS J 276(24);7319-28. PMID: 19909338

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."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

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

Herrington13: Herrington MB, Sitaras C (2013). "The influence of CsgD on the expression of genes of folate metabolism and hmp in Escherichia coli K-12." Arch Microbiol 195(8);559-69. PMID: 23824318

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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