Shan X, Wang L, Hoffmaster R, Kruger W D
Division of Population Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
J Biol Chem. 1999 Nov 12;274(46):32613-8. doi: 10.1074/jbc.274.46.32613.
Human methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-Methyltetrahydrofolate is a major methyl donor in the remethylation of homocysteine to methionine. Impaired MTHFR can cause high levels of homocysteine in plasma, which is an independent risk factor for vascular disease and neural tube defects. We have functionally characterized wild-type and several mutant alleles of human MTHFR in yeast, Saccharomyces cerevisiae. We have shown that yeast MET11 is a functional homologue of human MTHFR. Expression 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 auxotrophic phenotype in vivo. To understand the domain structure of human MTHFR, we have truncated the C terminus (50%) of the protein and demonstrated that expressing an N-terminal human MTHFR in met11(-) yeast cells rescues the growth phenotype, indicating that this region contains the catalytic domain of 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 MTHFR deficiency and two common missense polymorphisms found at high frequency in the general population. Three of the four missense mutations are unable to complement the auxotrophic phenotype of met11(-) yeast cells and show less than 7% enzyme activity of the wild type in vitro. Both of the two common polymorphisms are able to complement the growth phenotype, although one exhibited thermolabile enzyme activity in vitro. These results shall be useful for the functional characterization of MTHFR mutations and analysis structure/function relationship of the enzyme.
人类亚甲基四氢叶酸还原酶（MTHFR，EC 1.5.1.20）催化5,10-亚甲基四氢叶酸还原为5-甲基四氢叶酸。5-甲基四氢叶酸是同型半胱氨酸再甲基化生成甲硫氨酸过程中的主要甲基供体。MTHFR功能受损可导致血浆中同型半胱氨酸水平升高，这是血管疾病和神经管缺陷的独立危险因素。我们在酿酒酵母中对人类MTHFR的野生型和几个突变等位基因进行了功能表征。我们已经证明酵母MET11是人类MTHFR的功能同源物。在缺失MET11的酵母菌株中表达人类MTHFR cDNA可以在体外恢复该菌株的MTHFR活性，并在体内补充其甲硫氨酸营养缺陷型表型。为了了解人类MTHFR的结构域结构，我们截短了该蛋白的C末端（50%），并证明在met11（-）酵母细胞中表达N末端的人类MTHFR可以挽救生长表型，表明该区域包含该酶的催化结构域。然而，截短导致蛋白质水平降低，表明C末端可能对蛋白质稳定很重要。我们还对从严重MTHFR缺乏患者中鉴定出的四个错义突变和在普通人群中高频发现的两个常见错义多态性进行了功能表征。四个错义突变中的三个无法补充met11（-）酵母细胞的营养缺陷型表型，并且在体外显示出不到野生型7%的酶活性。两个常见多态性都能够补充生长表型，尽管其中一个在体外表现出热不稳定的酶活性。这些结果将有助于MTHFR突变的功能表征以及该酶的结构/功能关系分析。
