Small Molecule Rescue and Glycosidic Conformational Analysis of the Twister Ribozyme.
Small Molecule Rescue and Glycosidic Conformational Analysis of the Twister Ribozyme.
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DOI：
10.1021/acs.biochem.9b00742
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发表时间：
2019-11
期刊：
影响因子：
2.9
通讯作者：
Kyle J. Messina;R. Kierzek;Matthew A Tracey;P. Bevilacqua
中科院分区：
文献类型：
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作者：
Kyle J. Messina;R. Kierzek;Matthew A Tracey;P. Bevilacqua
近年来，自切割核酶的数量急剧增加，从而产生了四种已知的核酶催化策略，即α，β，γ和δ。一种这样的延伸被扭曲核酶利用，它被假设通过在生物pH下的缓冲催化和在较低的pH下直接通过合成腺嘌呤+1核苷酸碱基的N3间接地进行δ或一般的酸催化。在这里，我们通过化学拯救来检验A1的δ催化作用，并通过构象分析来检验核苷酸碱基的同步性的催化相关性。使用带有A1(N3)deaza或A1基础修饰的受抑扭曲核酶变体，我们观察到在咪唑和组氨酸等可质子化生物小分子存在的情况下，化学救援效果是原来的100倍，类似于先前报道的HDV核酶中C75U/C76Δ的救援值。Twister变体的Brnsted曲线图支持一个模型，在该模型中，小分子通过质子转移机制恢复催化活性，这表明野生型A1参与了质子转移，很可能是一般的酸催化。此外，在合适的溴原子背景下，通过糖苷构象分析，我们观察到8BrA1修饰的扭曲核酶比未修饰的A1核酶快10倍，支持结晶学数据，表明A1在进行质子转移时是同步的。总之，本研究提供的功能证据表明，紧邻切割位点下游的核苷酸在占据syn构象的同时，直接或间接参与了twist核酶中的一般酸碱催化作用。
The number of self-cleaving ribozymes has increased sharply in recent years, giving rise to elaborations of the four known ribozyme catalytic strategies, α, β, γ, and δ. One such extension is utilized by the twister ribozyme, which is hypothesized to conduct δ, or general acid catalysis, via N3 of the syn adenine +1 nucleobase indirectly via buffer catalysis at biological pH and directly at lower pH. Herein, we test the δ catalysis role of A1 via chemical rescue and the catalytic relevance of the syn orientation of the nucleobase by conformational analysis. Using inhibited twister ribozyme variants with A1(N3) deaza or A1 abasic modifications, we observe >100-fold chemical rescue effects in the presence of protonatable biological small molecules such as imidazole and histidine, similar to observed rescue values previously reported for C75U/C76Δ in the HDV ribozyme. Brønsted plots for the twister variants support a model in which small molecules rescue catalytic activity via a proton transfer mechanism, suggesting that A1 in the wild type is involved in proton transfer, most likely general acid catalysis. Additionally, through glycosidic conformational analysis in an appropriate background that accommodates the bromine atom, we observe that an 8BrA1-modified twister ribozyme is up to 10-fold faster than a nonmodified A1 ribozyme, supporting crystallographic data that show that A1 is syn when conducting proton transfer. Overall, this study provides functional evidence that the nucleotide immediately downstream of the cleavage site participates directly or indirectly in general acid-base catalysis in the twister ribozyme while occupying the syn conformation.