PSEUDOURIDINE SYNTHASES

在所有生物体中发现的假尿苷脱氨酶催化RNA中特定尿苷残基异构化为假尿苷。 假尿苷的特殊生理作用最近也出现了：1）假尿苷在U2 snRNA中是U2 snRNP的功能组装所必需的，所述U2 snRNP剪接前mRNA; 2）先天性角化不良疾病是由于缺乏与假尿苷合酶TruB具有序列相似性的蛋白质引起的; 3）假尿苷脱氨酶的抑制部分地解释了5-氟尿嘧啶的细胞毒性，5-氟尿嘧啶是一种在体内转化为5-氟尿嘧啶并掺入RNA的抗癌药物。提出了两种假尿苷脱氨酶TruB和RluA的机理研究。 通过整合定点诱变、化学修饰、动力学同位素效应的测定以及使用NMR方法直接观察假尿苷二磷酸酶和含有5-氟尿苷的RNA之间的加合物所提供的信息，将阐明活性位点处的事件。 定点诱变揭示了天冬氨酸的催化重要性，而位点特异性化学修饰将探测该关键天冬氨酸所起的催化作用。 动力学同位素效应将描述受该氨基酸影响的反应步骤。将测定含有5-氟尿苷的RNA与突变蛋白之间的加合物的化学结构，并与野生型酶的加合物进行比较。 这种比较将通过识别参与相互作用的蛋白质和RNA中的特定基团来揭示蛋白质-RNA相互作用的性质，这对于理解假尿苷合酶催化和5-氟尿嘧啶的作用至关重要。这些研究将扩大到研究更多的氨基酸残基参与催化，包括半胱氨酸残基和氨基酸的TruB对齐的蛋白质，其缺乏导致先天性角化不良。 总之，从这些不同的技术的观察加起来超过其部分的总和，结合起来，以产生一个更全面的了解在假尿苷酶的活性位点的事件，酶的生理重要性才刚刚开始得到充分认识。

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.