Friedt Jenna, Leavens Fern M V, Mercier Evan, Wieden Hans-Joachim, Kothe Ute
Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge AB T1K 3M4, Canada.
Nucleic Acids Res. 2014 Apr;42(6):3857-70. doi: 10.1093/nar/gkt1331. Epub 2013 Dec 26.
Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB's catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.
假尿苷合酶催化最常见的RNA修饰，并且可能采用相同的催化机制。除了催化性天冬氨酸残基外，其他残基对假尿苷形成催化作用的贡献尚不清楚。在这里，我们利用靶向tRNA中U55的细菌假尿苷合酶TruB，测试了活性位点中一个保守碱性残基在催化中的作用。将精氨酸181替换为赖氨酸会导致TruB的催化速率降低2500倍，而不影响tRNA结合。此外，我们分析了在所有TruB酶中保守且与tRNA的C56相互作用的第二壳层天冬氨酸残基（D90）的功能。定点诱变、生化和动力学研究表明，该残基对底物结合并不关键，但对催化有显著影响，因为用谷氨酸或天冬酰胺替换D90分别使催化速率降低30倍和50倍。与TruB野生型和TruB D90N的分子动力学模拟结果一致，我们提出了一个由催化性天冬氨酸（D48）、R181和D90组成的静电网络，该网络通过微调D48 - R181相互作用对催化作用很重要。在许多假尿苷合酶中发现了与D90类似的保守带负电荷残基，这表明这可能是一种普遍机制。
