Elizabeth E. Trimmer

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

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.

Mechanistic Studies of an Amine Oxidase Derived from d -Amino Acid Oxidase

The flavoprotein d-amino acid oxidase has long served as a paradigm for understanding the mechanism of oxidation of amino acids by flavoproteins. Recently, a mutant d-amino acid oxidase (Y228L/R283G) that catalyzed the oxidation of amines rather than amino acids was described [Yasukawa, K., et al. (2014) Angew. Chem., Int. Ed. 53, 4428-4431]. We describe here the use of pH and kinetic isotope effects with (R)-α-methylbenzylamine as a substrate to determine whether the mutant enzyme utilizes the same catalytic mechanism as the wild-type enzyme. The effects of pH on the steady-state and rapid-reaction kinetics establish that the neutral amine is the substrate, while an active-site residue, likely Tyr224, must be uncharged for productive binding. There is no solvent isotope effect on the kcat/Km value for the amine, consistent with the neutral amine being the substrate. The deuterium isotope effect on the kcat/Km value is pH-independent, with an average value of 5.3, similar to values found with amino acids as substrates for the wild-type enzyme and establishing that there is no commitment to catalysis with this substrate. The kcat/KO2 value is similar to that seen with amino acids as the substrate, consistent with the oxidative half-reaction being unperturbed by the mutation and with flavin oxidation preceding product release. All of the data are consistent with the mutant enzyme utilizing the same mechanism as the wild-type enzyme, transfer of hydride from the neutral amine to the flavin.

A role for glutamine 183 in the folate oxidative half-reaction of methylenetetrahydrofolate reductase from Escherichia coli

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes a ping-pong reaction with NADH and 5,10-methylenetetrahydrofolate (CH2-H4folate) to produce NAD+ and 5-methyltetrahydrofolate (CH3-H4folate). This work focuses on the function of the invariant, active-site aminoacyl residue Gln183. X-ray structures of the enzyme complexes Ered(wild-type)•NADH and Eox(Glu28Gln)•CH3-H4folate indicate that Gln183 makes key hydrogen-bonding interactions with both NADH and folate in their respective half-reactions, suggesting roles in binding each substrate. We propose that the polarity of Gln183 may also aid in stabilizing the proposed 5-iminium cation intermediate during catalysis in the oxidative half-reaction with folate. We have prepared mutants Gln183Ala and Gln183Glu, which we hypothesize to have altered charge/polarity and hydrogen bonding properties. We have examined the enzymes by steady-state and stopped-flow kinetics and by measurement of the flavin redox potentials. In the reductive half-reaction, NADH binding affinity and the rate of flavin reduction have not been hindered by either mutation. By contrast, our results support a minor role for Gln183 in the oxidative half-reaction. The Gln183Ala variant exhibited a 6-10 fold lower rate of folate reduction and bound CH2-H4folate with 7-fold lower affinity, whereas the Gln183Glu mutant displayed catalytic constants within 3-fold of the wild-type enzyme.