Functional characterization of human methylenetetrahydrofolate reductase in Saccharomyces cerevisiae

Human methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) catalyzes th e reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-Methyltetrahydrofolate is a major methyl donor in the remethylation of ho mocysteine to methionine, Impaired MTHFR can cause high levels of homocyste ine in plasma, which is an independent risk factor for vascular disease and neural tube defects. We have functionally characterized wildtype and sever al mutant alleles of human MTHFR in yeast, Saccharomyces cerevisiae. We hav e shown that yeast MET11 is a functional homologue of human MTHFR. Expressi on of the human MTHFR cDNA in a yeast strain deleted for MET11 can restore the strain's MTHFR activity in vitro and complement its methionine auxotrop hic phenotype in vivo, To understand the domain structure of human MTHFR, w e have truncated the C terminus (50%) of the protein and demonstrated that expressing an N-terminal human MTHFR in met11(-) yeast cells rescues the gr owth phenotype, indicating that this region contains the catalytic domain o f the enzyme. However, the truncation leads to the reduced protein levels, suggesting that the C terminus may be important for protein stabilization, We have also functionally characterized four missense mutations identified from patients with severe MTHFB deficiency and two common missense polymorp hisms found at high frequency in the general population, Three of the four missense mutations are unable to complement the auxotrophic phenotype of me t11(-) yeast cells and show less than 7% enzyme activity of the wild type i n vitro, Both of the two common polymorphisms are able to complement the gr owth phenotype, although one exhibited thermolabile enzyme activity in vitr o. These results shall be useful for the functional characterization of MTH FR mutations and analysis structure/function relationship of the enzyme.