Structural perturbations in the Ala → Val polymorphism of methylenetetrahydrofolate reductase:: How binding of folates may protect against inactivation
Structural perturbations in the Ala → Val polymorphism of methylenetetrahydrofolate reductase:: How binding of folates may protect against inactivation
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DOI：
10.1021/bi052294c
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发表时间：
2006-04-18
期刊：
影响因子：
2.9
通讯作者：
Ludwig, ML
中科院分区：
文献类型：
--
作者：
Pejchal, R;Campbell, E;Ludwig, ML
在人类亚甲基四氢叶酸还原酶(MTHFR)中，Ala222Val(677C->；T)多态编码一种不耐热的基因产物，该基因产物与同型半胱氨酸水平升高有关，并可能与心血管疾病的风险有关。在大肠杆菌MTHFR中产生等同的Ala到Val突变，与人类酶的催化域有30%的相同，创造了一种具有增强的耐热性的蛋白质。在人类和大肠杆菌MTHFR中，A-和GT；V突变增加了FAD的解离速度，并且在这两种酶中，FAD的丢失与第四级结构的变化有关[Yamada，K.，Chen，Z.，Rozen，R.，和Matthews，R.G.(2001)Proc。不是。阿卡德。SCI。美国，98,14853-14858；Guenther，B.D.，Seppard，C.A.，Tran，P.，Rozen，R.，Matthews，R.G.，and Ludwig，M.L.(1999年)NAT。结构。比奥尔。6,359-365]。叶酸已被证明可以保护人类和细菌的酶不受FAD的损失。尽管A->；V突变对FAD的亲和力有影响，但它位于催化结构域(βα)(8)桶的底部，位置不接触结合的FAD假体基团。在这里，我们报道了大肠杆菌MTHFR的Ala177Val突变体及其与5，10-二氮唑啉类似物LY309887的复合体的结构，并提出了该突变可能扰乱FAD结合的机制。螺旋5紧接在携带突变的环之前，携带几个与FAD相互作用的残基，包括Asn168、Arg171和Lys172。在突变酶的结构中，这个螺旋被移位，扰乱了蛋白质与FAD的相互作用。在与LY309887的复合体中，类似物的蝶呤样环堆叠在黄素的表面，并通过氢键与毗邻该表面的残基Gln183和Asp120结合。结合叶酸与辅因子的直接相互作用为FAD结合和叶酸结合之间的联系提供了一种机制，这在一定程度上解释了叶酸的保护作用。叶酸结合引起的构象变化也可能抑制FAD的解离。
In human methylenetetrahydrofolate reductase (MTHFR) the Ala222Val (677C -> T) polymorphism encodes a heat-labile gene product that is associated with elevated levels of homocysteine and possibly with risk for cardiovascular disease. Generation of the equivalent Ala to Val mutation in Escherichia coli MTHFR, which is 30% identical to the catalytic domain of the human enzyme, creates a protein with enhanced thermolability. In both human and E. coli MTHFR, the A -> V mutation increases the rate of dissociation of FAD, and in both enzymes, loss of FAD is linked to changes in quaternary structure [Yamada, K., Chen, Z., Rozen, R., and Matthews, R. G. (2001) Proc. Nad. Acad. Sci. U.S.A. 98, 14853-14858; Guenther, B. D., Sheppard, C. A., Tran, P., Rozen, R., Matthews, R. G., and Ludwig, M. L. (1999) Nat. Struct. Biol. 6, 359-365]. Folates have been shown to protect both human and bacterial enzymes from loss of FAD. Despite its effect on affinity for FAD, the A -> V mutation is located at the bottom of the (beta alpha)(8) barrel of the catalytic domain in a position that does not contact the bound FAD prosthetic group. Here we report the structures of the Ala177Val mutant of E. coli MTHFR and of its complex with the 5,10-dideazafolate analogue, LY309887, and suggest mechanisms by which the mutation may perturb FAD binding. Helix alpha 5, which immediately precedes the loop bearing the mutation, carries several residues that interact with FAD, including Asn168, Arg171, and Lys172. In the structures of the mutant enzyme this helix is displaced, perturbing protein-FAD interactions. In the complex with LY309887, the pterin-like ring of the analogue stacks against the si face of the flavin and is secured by hydrogen bonds to residues Gln183 and Asp120 that adjoin this face. The direct interactions of bound folate with the cofactor provide one mechanism for linkage between binding of FAD and folate binding that could account in part for the protective action of folates. Conformation changes induced by folate binding may also suppress dissociation of FAD.