The reaction mechanism and base recognition of RNase Rh

在本研究中，我们测定了RNase Rh的衍生物RNAP-Rh及其突变酶(RNAP-Rh、RNAP-Rh+2‘-AMP复合体、RNAP-Rh+2’-GMP复合体、Y57W、Y57W+2‘-AMP复合体、Y57W+3’-AMP复合体和Y57W+2‘-GMP复合体)的7种晶体结构。晶体的衍射数据是用振荡相机或魏森伯格相机收集的。根据RNaseRh的原子坐标确定了晶体结构，并用X-PLOR程序进行了精修，精修成功，所有的R值都覆盖到0.20以下。(1)发现该酶的碱基识别位点由Trp49、Tyr57和Asp51组成。底物的碱基夹在前两个残基之间，氢键连接到Asp51的侧链上。(2)该酶是腺嘌呤优先型核糖核酸酶，但我们可以通过浸泡技术将2‘-GMP引入核糖核酸酶RNAP-Rh和Y57W的晶体中。鸟嘌呤碱与腺嘌呤的结合方式相似，但也存在一定的差异。

During the present study we have determined seven crystal structures of RNase RNAP-Rh, a derivative of RNase Rh, and its mutant enzymes (RNAP-Rh, RNAP-Rh+2'-AMP complex, RNAP-Rh+2'-GMP complex, Y57W, Y57W+2'-AMP complex, Y57W+3'-AMP complex and Y57W+2'-GMP complex) . Diffraction data of the crystals were collected using an oscillation camera or a Weissenberg camera. The crystal structures were determined based on the atomic coordinates of RNase Rh, and refined usingthe program X-PLOR.The refinements were done successfully, and all the R-values were coverged to under 0.20. (1) We found out that the base recognition site of this enzyme consists of Trp49, Tyr57 and Asp51. The base of substrate is sandwiched in between the two former residues, and hydrogen-boned to the side chain of Asp51. (2) This enzyme is an adenine preferential RNase, but we could introduce 2'-GMP into the crystals of RNase RNAP-Rh and Y57W by soaking technique. The binding mode of guanine base is similar to that of adenine, but there are some differences between them.