PSEUDOURIDINE SYNTHASES

假尿苷合酶

• 批准号: 6879916
• 负责人: EUGENE G MUELLER
• 金额: $ 5.03万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879916
• 关键词: 5 fluorouridine; RNA binding protein; active sites; adduct; aspartate; carbon oxygen lyase; cysteine; enzyme mechanism; fluorouracil; isomerase; nuclear magnetic resonance spectroscopy; site directed mutagenesis; small nuclear RNA; tritium; uracil nucleoside

The pseudouridine synthases, found in all organisms, catalyze the isomerization of particular uridine residues in RNA to pseudouridine. Specific physiological roles for pseudouridine have also recently emerged: 1) pseudouridine is required in U2 snRNA for functional assembly of the U2 snRNP, which splices pre- mRNA; 2) the disease dyskeratosis congenita arises from the absence of a protein with sequence similarity to one pseudouridine synthase, TruB; 3) inhibition of pseudouridine synthases partly accounts for the cytotoxicity of 5-fluorouracil, an anti-cancer drug that is converted to 5-fluorouridine and incorporated into RNA in vivo. The mechanistic investigation of two pseudouridine synthases, TruB and RluA, is proposed. Events at the active site will be elucidated by integration of the information provided by site- directed mutagenesis, chemical modification, determination of kinetic isotope effects, and the direct observation of the adduct between pseudouridine synthases and RNA containing 5- fluorouridine using NMR methods. Site-directed mutagenesis revealed the catalytic importance of an aspartic acid, and site- specific chemical modification will probe the catalytic role played by that critical aspartic acid. Kinetic isotope effects will delineate the reaction step(s) affected by this amino acid. The chemical structure of the adduct between RNA containing 5- fluorouridine and the mutant protein will be determined and compared to that with the wild-type enzyme. This comparison will reveal the nature of protein-RNA interaction by identifying the particular groups in both the protein and the RNA that participate in the interaction, which is critical for understanding both pseudouridine synthase catalysis and the action of 5-fluorouracil. These studies will be expanded to examine more amino acid residues implicated in catalysis, including cysteine residues and the amino acids of TruB that align with the protein whose absence leads to dyskeratosis congenita. Taken together, the observations from these diverse techniques add up to more than the sum of their parts, combining to generate a fuller understanding of events at the active sites of pseudouridine synthases, enzymes whose physiological importance is just beginning to be fully appreciated.

在所有生物体中发现的假喹啉合酶可催化RNA中特定的尿苷残基与伪嘧啶的异构化。 假氨酸的特定生理作用最近也出现了：1）u2 snRNA中需要伪丁氨酸，以使U2 snRNP的功能组装剪接前mRNA； 2）疾病性脑膜病康涅狄格州源自缺乏与一种伪苷合酶Trub具有序列相似性的蛋白质； 3）抑制假氨酸合酶部分解释了5-氟尿嘧啶的细胞毒性，这是一种抗癌药物，转化为5-氟岛，并在体内掺入RNA中。提出了两种假喹啉合酶TRUB和RLUA的机械研究。 通过将定向的诱变，化学修饰，动力学同位素效应的测定以及使用NMR方法的直接观察，使用定位的诱变，化学修饰，测定动力学同位素效应，并直接观察到活跃位点的事件，并通过定位定向诱变，化学修饰，测定动力学同位素效应的确定以及直接观察含有5-氟尿苷的RNA之间的加合物。 定点诱变揭示了天冬氨酸的催化重要性，特异性化学修饰将探测该临界天冬氨酸所起的催化作用。 动力学同位素效应将描绘该氨基酸影响的反应步骤。将确定含有5-氟氨酸和突变蛋白的RNA之间的加合物的化学结构，并与野生型酶进行比较。 该比较将通过鉴定参与相互作用的蛋白质和RNA中的特定组来揭示蛋白-RNA相互作用的性质，这对于了解假喹啉合酶催化和5-氟尿嘧啶的作用至关重要。这些研究将扩展，以检查与催化有关的更多氨基酸残基，包括半胱氨酸残基和与蛋白质相一致的TRUB的氨基酸，其缺失导致患病障碍。 综上所述，这些多样化技术的观察结果加起来不仅仅是其各个部分的总和，从而使人们对假氨酸合成酶的活跃部位的事件有更深入的了解，即生理意义的酶刚刚开始充分理解。