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PDBsum entry 3fst
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Oxidoreductase
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PDB id
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3fst
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References listed in PDB file
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Key reference
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Title
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Functional role for the conformationally mobile phenylalanine 223 in the reaction of methylenetetrahydrofolate reductase from escherichia coli.
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Authors
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M.N.Lee,
D.Takawira,
A.P.Nikolova,
D.P.Ballou,
V.C.Furtado,
N.L.Phung,
B.R.Still,
M.K.Thorstad,
J.J.Tanner,
E.E.Trimmer.
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Ref.
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Biochemistry, 2009,
48,
7673-7685.
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PubMed id
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Abstract
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The flavoprotein methylenetetrahydrofolate reductase from Escherichia coli
catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) by
NADH via a ping-pong reaction mechanism. Structures of the reduced enzyme in
complex with NADH and of the oxidized Glu28Gln enzyme in complex with
CH(3)-H(4)folate [Pejchal, R., Sargeant, R., and Ludwig, M. L. (2005)
Biochemistry 44, 11447-11457] have revealed Phe223 as a conformationally mobile
active site residue. 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 to stack against the p-aminobenzoate ring of the folate.
Although Phe223 contacts each substrate in E. coli MTHFR, this residue is not
invariant; for example, a leucine occurs at this site in the human enzyme. To
examine the role of Phe223 in substrate binding and catalysis, we have
constructed mutants Phe223Ala and Phe223Leu. As predicted, our results indicate
that Phe223 participates in the binding of both substrates. The Phe223Ala
mutation impairs NADH and CH(2)-H(4)folate binding each 40-fold yet slows
catalysis of both half-reactions less than 2-fold. Affinity for CH(2)-H(4)folate
is unaffected by the Phe223Leu mutation, and the variant catalyzes the oxidative
half-reaction 3-fold faster than the wild-type enzyme. Structures of ligand-free
Phe223Leu and Phe223Leu/Glu28Gln MTHFR in complex with CH(3)-H(4)folate have
been determined at 1.65 and 1.70 A resolution, respectively. The structures show
that the folate is bound in a catalytically competent conformation, and Leu223
undergoes a conformational change similar to that observed for Phe223 in the
Glu28Gln-CH(3)-H(4)folate structure. Taken together, our results suggest that
Leu may be a suitable replacement for Phe223 in the oxidative half-reaction of
E. coli MTHFR.
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