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首页> 外文期刊>Journal of biochemical and molecular toxicology >Crystal structures of thymidylate synthase mutant R166Q: structural basis for the nearly complete loss of catalytic activity.
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Crystal structures of thymidylate synthase mutant R166Q: structural basis for the nearly complete loss of catalytic activity.

机译：胸苷酸合酶突变体R166Q的晶体结构：催化活性几乎完全丧失的结构基础。

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摘要

Thymidylate synthase (TS) catalyzes the folate-dependent methylation of deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP). We have investigated the role of invariant arginine 166, one of four arginines that contact the dUMP phosphate, using site-directed mutagenesis, X-ray crystallography, and TS from Escherichia coli. The R166Q mutant was crystallized in the presence of dUMP and a structure determined to 2.9 A resolution, but neither the ligand nor the sulfate from the crystallization buffer was found in the active site. A second structure determined with crystals prepared in the presence of dUMP and the antifolate 10-propargyl-5,8-dideazafolate revealed that the inhibitor was bound in an extended, nonproductive conformation, partially occupying the nucleotide-binding site. A sulfate ion, rather than dUMP, was found in the nucleotide phosphate-binding site. Previous studies have shown that the substitution at three of the four arginines of the dUMP phosphate-binding site is permissive; however; for Arg166, all the mutations lead to a near-inactive mutant. The present structures of TS R166Q reveal that the phosphate-binding site is largely intact, but with a substantially reduced affinity for phosphate, despite the presence of the three remaining arginines. The position of Cys146, which initiates catalysis, is shifted in the mutant and resides in a position that interferes with the binding of the dUMP pyrimidine moiety.
机译：胸苷酸合酶（TS）催化脱氧尿苷单磷酸（dUMP）的叶酸依赖性甲基化反应，形成胸苷单磷酸（dTMP）。我们使用定点诱变，X射线晶体学和大肠杆菌的TS，研究了与dUMP磷酸酯接触的四个精氨酸不变的精氨酸166的作用。 R166Q突变体在dUMP存在下结晶，结构确定为2.9 A拆分，但在活性位点中未发现配体或结晶缓冲液中的硫酸盐。用在dUMP和抗叶酸剂10-炔丙基-5,8-二氮杂叶酸存在下制备的晶体确定的第二种结构表明抑制剂以扩展的非生产性构象结合，部分占据了核苷酸结合位点。在核苷酸磷酸结合位点发现了硫酸根离子，而不是dUMP。先前的研究表明，在dUMP磷酸结合位点的四个精氨酸中的三个被取代是允许的。然而;对于Arg166，所有突变均导致近乎失活的突变。 TS R166Q的当前结构显示，尽管存在三个剩余的精氨酸，但磷酸盐结合位点基本上是完整的，但与磷酸盐的亲和力却大大降低。启动催化的Cys146的位置在突变体中转移，并且位于干扰dUMP嘧啶部分结合的位置。

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