Methylenetetrahydrofolate reductase [NAD(P)H]

 

E. coli methylenetetrahydrofolate reductase (MTHFR) catalyses the NADH-dependent reduction of 5,10-methylenetetrahydrofolate (CH2-H4folate) to 5 methyltetrahydrofolate (CH3-H4folate) using the cofactor flavin adenine dinucleotide (FAD) as an intermediate hydride acceptor and donor. MTHFR is the only route of CH3-H4folate which is used by methionine synthase to convert homocysteine to methionine.

E. coli MTHFR is a homotetramer that dissociates into dimers on dilution. Its catalytic domain is a (beta-alpha)8 barrel. Bacterial MTHFR enzymes are simpler than mammalian MTHFR in that the catatlytic domain constitutes the entire sequence, with no regulatory sequence present.

 

Reference Protein and Structure

Sequence
P0AEZ1 UniProt (1.5.1.20) IPR004620 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1zp3 - E. coli Methylenetetrahydrofolate Reductase (oxidized) (1.85 Å) PDBe PDBsum 1zp3
Catalytic CATH Domains
3.20.20.220 CATHdb (see all for 1zp3)
Cofactors
Fadh2(2-) (1), Water (2)
Click To Show Structure

Enzyme Reaction (EC:1.5.1.20)

(6R)-5,10-methylenetetrahydrofolate(2-)
CHEBI:15636ChEBI
+
hydron
CHEBI:15378ChEBI
+
NADPH(4-)
CHEBI:57783ChEBI
→
5-methyltetrahydrofolate(2-)
CHEBI:18608ChEBI
+
NADP(3-)
CHEBI:58349ChEBI
Alternative enzyme names: 5,10-CH(2)-H(4)folate reductase, 5,10-methylenetetrahydrofolate reductase (NADPH), 5,10-methylenetetrahydrofolic acid reductase, Methylenetetrahydrofolate (reduced nicotinamide adenine dinucleotide phosphate) reductase, 5,10-methylenetetrahydrofolate reductase (FADH(2)), 5,10-methylenetetrahydrofolate reductase, 5,10-methylenetetrahydropteroylglutamate reductase, 5-methyltetrahydrofolate:(acceptor) oxidoreductase, 5-methyltetrahydrofolate:NAD oxidoreductase, 5-methyltetrahydrofolate:NAD(+) oxidoreductase, 5-methyltetrahydrofolate:NADP(+) oxidoreductase, Methylenetetrahydrofolate (reduced riboflavin adenine dinucleotide) reductase, Methylenetetrahydrofolate reductase, Methylenetetrahydrofolate reductase (NADPH), Methylenetetrahydrofolate reductase (NADPH(2)), Methylenetetrahydrofolic acid reductase, N(5,10)-methylenetetrahydrofolate reductase, N(5),N(10)-methylenetetrahydrofolate reductase, MetF, MTHFR,

Enzyme Mechanism

Introduction

E. coli MTHFR catalysis proceeds by a ping pong Bi-Bi reaction mechanism. The enzyme catalyses individual half-reactions, with the reduction of enzyme-bound FAD by NADH to form NAD+, which is released prior to CH2-H4folate binding. CH2-H4folate is thought to be converted to CH3-H4folate by reduced E-FAD via a 5-iminium cation intermediate.

NADH binds at the si face of E-FAD(ox) cofactor. The 4S-hydrogen of NADH is transferred as a hydride to N5 of FAD, forming E-FAD(red) and NAD+. NAD+ dissociates from the enzyme. CH2-H4folate also binds at the si face of FAD. N10 of CH2-H4folate is thought to be protonated by Glu28, which leads to the opening of the five-membered imidazolidine ring of CH2-H4folate to form the 5-iminium cation intermediate. The intermediate is stabilised by Asp120 and Glu28. A hydride is transferred from N5 of E-FAD(red) to the exocyclic methylene group (C11) of the stabilised 5-iminium cation intermediate to form the product CH3-H4folate.

Catalytic Residues Roles

UniProt PDB* (1zp3)
Phe223 Phe223A This conformationally mobile active site residue plays an important role in binding and positioning the NAD(P) and FAD molecules: In the NADH complex, the NADH adopts an unusual hairpin conformation and is wedged between the isoalloxazine ring of the FAD and the side chain of Phe223. In the folate complex, Phe223 swings out from its position in the NADH complex in order to stack against the p-aminobenzoate ring of the folate. steric locator
Glu28 Glu28A Glu28 has been implicated as the general acid that acts by protonating N10 of the folate leading to the opening of the five-membered imidazolidine ring of CH2-H4folate. This is thought to be coordinated by a water molecule, and Glu28 is likely to be reprotonated by a proton relay involving H273 and S26 from water in the bulk solvent. Glu28 is also involved in stabilising the 5-iminium cation. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
Asp120 Asp120A Asp120 has major role in folate binding and 5-iminium cation intermediate stabilisation. The carboxylate oxygens form bidentate hydrogen bonds with N3 and 2-amino groups of the pterin ring of both the substrate and the intermediate. The negative charge of Asp120 also has a minor role in modulating the reactivity of flavin. hydrogen bond acceptor, electrostatic stabiliser
Ser26, His273 Ser26A, His273A Forms a proton relay chain (with Glu28 and a water molecule). hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
*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

aromatic unimolecular elimination by the conjugate base, aromatic bimolecular nucleophilic addition, hydride transfer, proton transfer, overall reactant used, cofactor used, intermediate formation, overall product formed, intramolecular elimination, proton relay, decyclisation, aromatic bimolecular elimination, bimolecular nucleophilic addition, intermediate terminated, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Pejchal R et al. (2005), Biochemistry, 44, 11447-11457. Structures of NADH and CH3-H4Folate Complexes ofEscherichia coliMethylenetetrahydrofolate Reductase Reveal a Spartan Strategy for a Ping-Pong Reaction†,‡. DOI:10.1021/bi050533q. PMID:16114881.
  2. Lee MN et al. (2009), Biochemistry, 48, 7673-7685. Functional Role for the Conformationally Mobile Phenylalanine 223 in the Reaction of Methylenetetrahydrofolate Reductase fromEscherichia coli. DOI:10.1021/bi9007325. PMID:19610625.
  3. Pejchal R et al. (2006), Biochemistry, 45, 4808-4818. Structural Perturbations in the Ala → Val Polymorphism of Methylenetetrahydrofolate Reductase:  How Binding of Folates May Protect against Inactivation†,‡. DOI:10.1021/bi052294c. PMID:16605249.
  4. Trimmer EE et al. (2005), Biochemistry, 44, 6809-6822. Aspartate 120 ofEscherichia coliMethylenetetrahydrofolate Reductase:  Evidence for Major Roles in Folate Binding and Catalysis and a Minor Role in Flavin Reactivity†,‡. DOI:10.1021/bi0477236. PMID:15865426.
  5. Trimmer EE et al. (2001), Biochemistry, 40, 6216-6226. Folate Activation and Catalysis in Methylenetetrahydrofolate Reductase fromEscherichia coli:  Roles for Aspartate 120 and Glutamate 28†. DOI:10.1021/bi002790v. PMID:11371182.

Step 1. NAD(P) eliminates a hydride ion, which adds to FAD with concomitant deprotonation of water at the N1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp120A hydrogen bond acceptor, electrostatic stabiliser
Ser26A hydrogen bond acceptor, hydrogen bond donor
His273A hydrogen bond acceptor, hydrogen bond donor
Glu28A hydrogen bond acceptor, hydrogen bond donor
Phe223A steric locator

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, ingold: aromatic bimolecular nucleophilic addition, hydride transfer, proton transfer, overall reactant used, cofactor used, intermediate formation, overall product formed

Step 2. The substrate initiates the cleavage of the five-membered ring, forming the 5-iminium cation intermediate, with concomitant deprotonation of water through a proton relay chain of water, Glu28, His273 and Ser26.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp120A hydrogen bond acceptor, electrostatic stabiliser
Ser26A hydrogen bond acceptor, hydrogen bond donor, proton relay
His273A hydrogen bond acceptor, hydrogen bond donor, proton relay
Glu28A hydrogen bond acceptor, hydrogen bond donor, proton relay
Phe223A steric locator
Glu28A proton donor
His273A proton donor
Ser26A proton donor
His273A proton acceptor
Ser26A proton acceptor
Glu28A proton acceptor

Chemical Components

ingold: intramolecular elimination, proton transfer, overall reactant used, intermediate formation, proton relay, decyclisation

Step 3. Water deprotonates FAD, causing the elimination of a hydride which is added to the 5-iminium carbon of the intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp120A hydrogen bond acceptor, electrostatic stabiliser
Ser26A hydrogen bond acceptor, hydrogen bond donor
His273A hydrogen bond acceptor, hydrogen bond donor
Glu28A hydrogen bond acceptor, hydrogen bond donor
Phe223A steric locator

Chemical Components

ingold: aromatic bimolecular elimination, ingold: bimolecular nucleophilic addition, hydride transfer, intermediate terminated, native state of cofactor regenerated, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. NAD(P) eliminates a hydride ion, which adds to FAD with concomitant deprotonation of water at the N1.

Step 2. The substrate initiates the cleavage of the five-membered ring, forming the 5-iminium cation intermediate, with concomitant deprotonation of water through a proton relay chain of water, Glu28, His273 and Ser26.

Step 3. Water deprotonates FAD, causing the elimination of a hydride which is added to the 5-iminium carbon of the intermediate.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Alex Gutteridge, Craig Porter