A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.
A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.
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DOI：
10.1002/anie.201506601
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发表时间：
2015-12-07
期刊：
影响因子：
--
通讯作者：
Micura R
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文献类型：
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作者：
Košutić M;Neuner S;Ren A;Flür S;Wunderlich C;Mairhofer E;Vušurović N;Seikowski J;Breuker K;Höbartner C;Patel DJ;Kreutz C;Micura R
核酶催化磷酸二酯骨架的位点特异性裂解。据报道，扭曲核酶的最小版本缺乏系统发育上保守的茎P1，同时保持野生型活性。原子诱变表明，在裂解位点的腺嘌呤-6的氮原子N1和N3对裂解是必不可少的。通过核磁共振波谱测定，在twister核酶的这个位置上，13c2标记的腺嘌呤的pKa值为5.1，与单独在底物中的相同腺嘌呤的pKa相比，pKa值明显变化。这一发现指出了腺嘌呤-6在催化机制中的潜在作用，除了先前确定的不变鸟嘌呤-48和Mg2+离子，两者都直接配位到可裂磷酸盐的非桥接氧原子上；对于后者，更多的证据源于观察到Mn2+或Cd2+加速了磷酸化底物的裂解。2 ' -OCH3-U5修饰的扭曲核酶的新的2.6 Å x射线结构进一步强调了这种金属离子结合位点的相关性。
Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a 13C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg2+ ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn2+ or Cd2+ accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X-ray structure of a 2′-OCH3-U5 modified twister ribozyme.