Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Biochemistry. 2011 Jan 25;50(3):426-36. doi: 10.1021/bi101737z. Epub 2010 Dec 29.
RNA containing 5-fluorouridine (F(5)U) had previously been used to examine the mechanism of the pseudouridine synthase TruA, formerly known as pseudouridine synthase I [Gu et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 14270-14275]. From that work, it was reasonably concluded that the pseudouridine synthases proceed via a mechanism involving a Michael addition by an active site aspartic acid residue to the pyrimidine ring of uridine or F(5)U. Those conclusions rested on the assumption that the hydrate of F(5)U was obtained after digestion of the product RNA and that hydration resulted from hydrolysis of the ester intermediate between the aspartic acid residue and F(5)U. As reported here, (18)O labeling definitively demonstrates that ester hydrolysis does not give rise to the observed hydrated product and that digestion generates not the expected mononucleoside product but rather a dinucleotide between a hydrated isomer of F(5)U and the following nucleoside in RNA. The discovery that digestion products are dinucleotides accounts for the previously puzzling differences in the isolated products obtained following the action of the pseudouridine synthases TruB and RluA on F(5)U in RNA.
此前，含有 5-氟尿嘧啶核苷（F(5)U）的 RNA 已被用于研究假尿嘧啶核苷合酶 TruA（以前称为假尿嘧啶核苷合酶 I）的机制[Gu 等人。（1999）Proc. Natl. Acad. Sci. U.S.A. 96, 14270-14275]。从这项工作中，可以合理地得出结论，假尿嘧啶核苷合酶通过一种涉及活性部位天冬氨酸残基对尿嘧啶或 F(5)U 的嘧啶环进行迈克尔加成的机制进行。这些结论基于这样的假设，即 F(5)U 的水合物是在消化产物 RNA 后获得的，并且水合作用是由于天冬氨酸残基和 F(5)U 之间的酯中间产物的水解产生的。正如这里所报道的，（18）O 标记明确证明酯水解不会产生观察到的水合产物，并且消化不会产生预期的单核苷酸产物，而是在 F(5)U 的水合异构体和 RNA 中的下一个核苷之间形成二核苷酸。消化产物是二核苷酸的发现解释了之前在 TruB 和 RluA 在 RNA 上作用于 F(5)U 时获得的分离产物中出现的令人困惑的差异。
