Methionine synthase is one of the major enzymes that metabolizes homocysteine and catalyzes the transfer of a methyl group from the substrate, methyltetrahydrofolate to the cobalamin cofactor and subsequently, to the second substrate, homocysteine to form methionine. Its cofactor, methylcobalamin, cycles between different oxidation states in the catalytic cycle and is prone to oxidative inactivation. The dual flavoenzyme, methionine synthase reductase (MSR), transfers electrons from NADPH to the cobalamin cofactor and, in conjunction with S-adenosylmethionine, reactivates mammalian methionine synthase. The physiological importance of this reaction is in freeing methyltetrahydrofolate from the tetrahydrofolate pool to support DNA synthesis. The clinical significance of the methionine synthase-catalyzed reaction stems from hyperhomocysteinemia, representing a risk factor in the multifactorial etiology of cardiovascular diseases. While hereditary hyperhomocysteinemia is characterized by plasma homocysteine levels surging up to 100–200 μM, the levels in mild hyperhomocysteinemia generally varies between 15–40 μM. A detailed understanding of the regulation of homocysteine metabolism is of substantial biomedical interest. With this in mind, we embarked on unraveling the biochemical function and properties of human MSR, which had been previously described only at a genetic level. The objective was to evaluate the role of MSR in methionine synthase activation which had been previously suggested by genetic studies. We successfully expressed recombinant human MSR and characterized its spectral, kinetic and electron transfer properties. Importantly, we demonstrated for the first time, that MSR is able to activate methionine synthase in the presence of NADPH and S-adenosylmethionine. We have also subjected two common variants of MSR, I22M and S175L to similar biochemical scrutiny. The I22M polymorphism has been linked to hyperhomocysteinemia and homocysteine-related diseases, in conjunction with other genetic and nutritional factors. From a comparison of (i) the UV-visible and EPR spectroscopic properties, (ii) the electron transfer reaction to the natural and to artificial electron acceptors, and (iii) the redox midpoint potentials of the flavin cofactors of the MSR polymorphic variants with the wild-type enzyme, we have postulated that the polymorphisms result in decreased efficiency between the redox partners during methionine synthase activation
