Functional role for the conformationally mobile phenylalanine 223 in the reaction of methylenetetrahydrofolate reductase from Escherichia coli

Abstract

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.

FIGURE 1

Stereographic view of the wild-type MTHFR-NADH complex (yellow, pdb code 1zpt) and the Glu28Gln MTHFR-CH₃H₄folate complex (white, pdb code 1zp4) (23). The ligands occupy partially overlapping sites at the si face of the FAD and residues Asp120, Phe223, and Glu28 (mutated as Gln28) interact with both substrates. The L4 loop moves as a rigid unit to adopt two conformations with different orientations of Asp120. The aromatic side chain of Phe223 adjusts its position to either stack against the adenine ring of the NADH or against the pABA ring of the folate.

FIGURE 2

Spectra of purified Phe223 mutant and wild-type MTHFR enzymes. Phe223Leu, (), Phe223Ala, (), and wild-type, () enzymes (30 μM) in 50 mM potassium phosphate (pH 7.2) buffer containing 0.3 mM EDTA and 10% glycerol. For all enzymes, wavelength of maximal absorbance is 447 nm and molar extinction coefficient is 14,300 M⁻¹cm⁻¹.

FIGURE 3

Structures of Phe223Leu MTHFR. (A). Ribbon drawing of Phe223Leu MTHFR. The protein is colored in a rainbow scheme, with blue at the N-terminus and red at the C-terminus. The FAD is drawn as sticks in yellow. The side chain of Leu223 is shown in white. (B). Comparison of the active sites of Phe223Leu (magenta) and wild-type MTHFR (white, PDB code 1zp3). The FAD cofactors of both enzymes are colored yellow.

FIGURE 4

Reduction of the Phe223Leu enzyme by NADH. Oxidized enzyme, 10 μM, was mixed with solutions of 50 (), 100 (), 200 (), 300 (), 500 (), 800 (), 1500 (), and 2000 () μM NADH (concentrations after mixing). Stopped-flow reaction traces were monitored at 450 nm. The reaction was monophasic. (Inset) Dependence of the observed rate constant for reduction on NADH concentration. The data were fit to a hyperbolic equation (eq 4), which yielded an apparent K_d of 440 ± 20 μM for NADH and a maximum rate constant for reduction (k₂’, according to Scheme 2) of 35 ± 1 s⁻¹. Reaction conditions were 50 mM potassium phosphate (pH 7.2) buffer containing 0.3 mM EDTA and 10% glycerol at 25 °C.

FIGURE 5

Reoxidation of reduced Phe223Leu enzyme by CH₂-H₄folate. Photoreduced enzyme (10 μM) was mixed with solutions of 17 (), 25 (), 50 (), 100 (), 200 (), and 300 μM () CH₂-H₄folate (concentrations after mixing). Stopped-flow reaction traces, monitored at 450 nm, were monophasic. (Inset) Dependence of the observed rate constant on CH₂-H₄folate concentration. A fit of the data to eq 4 yielded an apparent K_d for CH₂-H₄folate of 9 ± 2 μM and a maximal rate constant for oxidation (k₅’, according to Scheme 3) of 34 ± 1 s⁻¹. Reaction conditions were 50 mM potassium phosphate (pH 7.2) buffer containing 0.3 mM EDTA and 10% glycerol at 25 °C.

FIGURE 6

Reoxidation of reduced Phe223Ala enzyme by CH₂-H₄folate. (A). Photoreduced enzyme (10 μM) was mixed with solutions of 50 (), 100 (), 190 (), 385 (), 580 (), and 980 () μM CH₂-H₄folate (concentrations after mixing). Stopped-flow reaction traces, monitored at 450 nm, were biphasic. The fast and slow phases accounted for 84%, and 16%, respectively, of the total observed absorbance change. (Inset). Dependence of the slow phase on CH₂-H₄folate concentration. A hyperbolic fit of the data gave an apparent K_d of 500 ± 230 μM for CH₂-H₄folate and a maximum rate constant of 4.5 ± 1.1 s⁻¹. Reaction conditions were 50 mM potassium phosphate (pH 7.2) buffer containing 0.3 mM EDTA and 10% glycerol at 25 °C.

FIGURE 7

Active site of the Phe223Leu/Glu28Gln-CH₃-H₄folate complex. (A) The active site with electron density. The FAD and folate of the complex are colored yellow and green, respectively. Selected side chains are drawn in white. The map is a simulated annealing σ_A-weighted F_o-F_c map contoured at 2σ. Prior to map calculation, the folate ligand was removed from the model and simulated annealing refinement was performed with PHENIX. (B) Superposition of the Phe223Leu/Glu28Gln-CH₃-H₄folate complex (magenta) and the Glu28Gln-CH₃-H₄folate complex (white, PDB code 1zp4). (C) Close-up view of the interactions between Leu223 and the folate in the Phe223Leu/Glu28Gln-CH₃-H₄folate complex. (D) Close-up view of the interactions between Phe223 and the folate in the Glu28Gln-CH₃-H₄folate complex (PDB code 1zp4).

FIGURE 8

Conformational changes induced by the binding of CH₃-H₄folate. The structure of the Phe223Leu/Glu28Gln-CH₃H₄folate complex is shown in white with yellow FAD and green folate. The side chains of Gln219 and Leu223 of the ligand-free Phe223Leu structure are shown in cyan with accompanying van der Waals dot surfaces. Note that these surfaces overlap with the CH₃-H₄folate.

Scheme 1

Proposed Mechanism for the Oxidative Half-Reaction

Scheme 2

Kinetic Mechanism for the Reductive Half-Reaction

Scheme 3

Kinetic Mechanism for the Oxidative Half-Reaction