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Catalytic Site Atlas

CSA LITERATURE entry for 2fmn

E.C. namemethylenetetrahydrofolate reductase [NAD(P)H]
SpeciesEscherichia coli (Bacteria)
E.C. Number (IntEnz) 1.5.1.20
CSA Homologues of 2fmn
CSA Entries With UniProtID P0AEZ1
CSA Entries With EC Number 1.5.1.20
PDBe Entry 2fmn
PDBSum Entry 2fmn
MACiE Entry M0120

Literature Report

IntroductionE. 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.
The E.C. code was previously 1.7.99.5, but it has been reclassified.
MechansimE. 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.
Reaction

Catalytic Sites for 2fmn

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
GluA2828macie:sideChainGlu28 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.
AspA120120macie:sideChainAsp120 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.

Annotated By Reference To The Literature - Site 2 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
GluB2828macie:sideChainGlu28 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.
AspB120120macie:sideChainAsp120 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.

Annotated By Reference To The Literature - Site 3 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
GluC2828macie:sideChainGlu28 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.
AspC120120macie:sideChainAsp120 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.

Literature References

Notes:
Pejchal R
Structural perturbations in the Ala --> Val polymorphism of methylenetetrahydrofolate reductase: how binding of folates may protect against inactivation.
Biochemistry 2006 45 4808-4818
PubMed: 16605249
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