Crystal structures of thymidylate synthase mutant R166Q: Structural basis for the nearly complete loss of catalytic activity
Crystal structures of thymidylate synthase mutant R166Q: Structural basis for the nearly complete loss of catalytic activity
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DOI：
10.1002/jbt.20122
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发表时间：
2006-01-01
影响因子：
3.6
通讯作者：
Montfort, WR
中科院分区：
文献类型：
--
作者：
Sotelo-Mundo, RR;Changchien, LM;Montfort, WR
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 研究了恒定精氨酸 166（接触 dUMP 磷酸盐的四种精氨酸之一）的作用。 R166Q 突变体在 dUMP 存在下结晶，结构分辨率为 2.9 埃，但在活性位点中未发现配体或结晶缓冲液中的硫酸盐。在 dUMP 和抗叶酸剂 10-炔丙基-5,8-二脱氮杂叶酸存在下制备的晶体测定的第二种结构表明，该抑制剂以延伸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中发现了硫酸根离子，而不是 dUMP。先前的研究表明，dUMP 磷酸结合位点的四个精氨酸中的三个的取代是允许的；然而;对于 Arg166，所有突变都会导致近乎失活的突变体。 TS R166Q 的现有结构表明，磷酸盐结合位点基本上是完整的，但尽管存在三个剩余的精氨酸，但对磷酸盐的亲和力却大大降低。启动催化作用的 Cys146 的位置在突变体中发生了移动，并且位于干扰 dUMP 嘧啶部分结合的位置。 (c) 2006 年 Wiley 期刊公司
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 angstrom 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. (c) 2006 Wiley Periodicals, Inc.