5-formyltetrahydrofolate cyclo-ligase

 

Tetrahydrofolates (THFs) are cofactors that donate or accept single carbon units in metabolic processes. 5,10-methenyltetrahydrofolate synthetase (MTHFS) is the only known enzyme to use 5-formylTHF. MTHFS catalyses tbe conversion of 5-formyTHF to 5,10-methenylTHF coupled to the hydrolysis of ATP to ADP in the presence of magnesium.
In solution, MTHFS exists 50% dimeric form, 50% in monomeric form.

 

Reference Protein and Structure

Sequence
P75430 UniProt (6.3.3.2) IPR002698 (Sequence Homologues) (PDB Homologues)
Biological species
Mycoplasma pneumoniae M129 (Bacteria) Uniprot
PDB
1u3f - Structural and Functional Characterization of a 5,10-Methenyltetrahydrofolate Synthetase from Mycoplasma pneumoniae (GI: 13508087) (2.5 Å) PDBe PDBsum 1u3f
Catalytic CATH Domains
3.40.50.10420 CATHdb (see all for 1u3f)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:6.3.3.2)

(6S)-5-formyltetrahydrofolate(2-)
CHEBI:57457ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
→
(6R)-5,10-methenyltetrahydrofolate
CHEBI:57455ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: 5,10-methenyltetrahydrofolate synthetase, 5-formyltetrahydrofolate cyclodehydrase, Formyltetrahydrofolic cyclodehydrase, Methenyl-THF synthetase, 5-Formyltetrahydrofolate cyclodehydrase,

Enzyme Mechanism

Introduction

The formyl oxygen of the major rotamer of the enolate form of 5-formylTHF makes a nucleophilic attack on the gamma-phosphoryl group of ATP to form a phosphorylated intermediate. At this intermediate, enzyme-bound ADP can freely equilibriate with the solvent ADP. The N10 of the iminium phosphate intermediate attacks the formyl carbon nucleophilically to form a putative tetrahedral intermediate. This intermediate then collapses to eliminate phosphate and form the product. There does not appear to be any direct involvement of residues in catalysis, although Arg115 is involved in increasing the electrophilicity of the ATP and/or the iminium phosphate intermediate. While not involved directly in catalysis the magnesium ion is essential for the function this enzyme. Aspartate residues 124 and 154 are in close proximity to the magnesium ion, which is in turn in close proximity to the alpha and beta phosphates of the ADP molecule. The aspartate residues are essential for coordinating the magnesium ion which is necessary to neutralize the negatively charged phosphate groups of ADP.

Catalytic Residues Roles

UniProt PDB* (1u3f)
Asp124, Asp154 Asp124(149)A, Asp154(179)A Binds Mg(II) ion. metal ligand
Arg115 Arg115(140)A The positively-charged side chain of Arg115 is positioned to draw electron density away from the phosphate in either its ATP and or its iminium intermediate form. Arg causes the phosphorus or iminium carbon to become more electrophilic, facilitating nucleophilic attack at these positions. increase electrophilicity, electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

keto-enol tautomerisation, overall reactant used, overall product formed, bimolecular nucleophilic substitution, proton transfer, intramolecular nucleophilic addition, cyclisation, intramolecular elimination

References

  1. Hancock AN et al. (2008), Protein J, 27, 303-308. Investigations of the Roles of Arginine 115 and Lysine 120 in the Active Site of 5,10-Methenyltetrahydrofolate Synthetase from Mycoplasma pneumoniae. DOI:10.1007/s10930-008-9138-z. PMID:18473156.
  2. Tolley M et al. (2012), Protein J, 31, 519-528. Investigations of amino acids in the ATP binding site of 5,10-methenyltetrahydrofolate synthetase. DOI:10.1007/s10930-012-9428-3. PMID:22773193.
  3. Huang T et al. (1995), J Biol Chem, 270, 22296-22300. Mechanism for the Coupling of ATP Hydrolysis to the Conversion of 5-Formyltetrahydrofolate to 5,10-Methenyltetrahydrofolate. DOI:10.1074/jbc.270.38.22296. PMID:7673211.

Step 1. The substrate tautomerizes into the enolate form

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Catalytic Residues Roles

Residue Roles
Arg115(140)A electrostatic stabiliser
Asp124(149)A metal ligand
Asp154(179)A metal ligand

Chemical Components

keto-enol tautomerisation

Step 2. The enolate oxygen performs a nucleophilic attack on the gamma-phosphate causing ADP to be displaced.

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Catalytic Residues Roles

Residue Roles
Arg115(140)A increase electrophilicity, electrostatic stabiliser
Asp124(149)A metal ligand
Asp154(179)A metal ligand

Chemical Components

overall reactant used, overall product formed, ingold: bimolecular nucleophilic substitution

Step 3. N10 attacks the intermediate forming a new tetrahedral intermediate.

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Catalytic Residues Roles

Residue Roles
Arg115(140)A increase electrophilicity, electrostatic stabiliser
Asp124(149)A metal ligand
Asp154(179)A metal ligand

Chemical Components

proton transfer, ingold: intramolecular nucleophilic addition, cyclisation

Step 4. The intermediate collapses and the product is formed.

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Catalytic Residues Roles

Residue Roles
Arg115(140)A electrostatic stabiliser
Asp124(149)A metal ligand
Asp154(179)A metal ligand

Chemical Components

ingold: intramolecular elimination, overall product formed
Download: Image, Marvin File

Step 1. The substrate tautomerizes into the enolate form

Step 2. The enolate oxygen performs a nucleophilic attack on the gamma-phosphate causing ADP to be displaced.

Step 3. N10 attacks the intermediate forming a new tetrahedral intermediate.

Step 4. The intermediate collapses and the product is formed.

Products.

Contributors

Gemma L. Holliday, James Willey