Vassylyev D G, Katayanagi K, Ishikawa K, Tsujimoto-Hirano M, Danno M, Pähler A, Matsumoto O, Matsushima M, Yoshida H, Morikawa K
Protein Engineering Research Institute, Osaka, Japan.
J Mol Biol. 1993 Apr 5;230(3):979-96. doi: 10.1006/jmbi.1993.1214.
RNase F1, a guanine-specific ribonuclease from Fusarium moniliforme, was crystallized in two different forms, in the absence of an inhibitor and in the presence of 2'GMP. The crystal structure of the RNase F1 free form was solved by the molecular replacement method, using the co-ordinates of the RNase T1 complex with 2'GMP, and was refined to a final R-factor of 18.7%, using the data extended to 1.3 A resolution. For the crystal structure of the RNase F1 complex with 2'GMP, the solution of the molecular replacement method was obtained on the basis of the co-ordinates of the RNase F1 free form, and was refined to a final R-factor of 16.8%, using the data up to 2 A resolution. The two crystal structures of the RNase F1 free form and the complex with 2'GMP are very similar to each other as reflected by a small root-mean-square displacement (r.m.s.d.) value of 0.43 A for all C alpha atoms. The main differences between the two structures are associated with binding of 2'GMP in the substrate recognition site in the loop between Tyr42 and Glu46. A structural comparison between RNase F1 and RNase T1 shows a substantial similarity between all the C alpha atoms, as evidenced by a r.m.s.d. value of 1.4 A. The loop from residues 32 to 38 was strikingly different between these two enzymes, in both its conformation and its hydrogen bonding schemes. The side-chain of a catalytically active residue, His92, is shifted away from the catalytic site in RNase F1 by 1.3 A and 0.85 A with respect to the corresponding positions in the RNase T1 free form and in the RNase T1 complex with 2'GMP, respectively. In the RNase F1 complex, the guanine base of 2'GMP has a syn conformation about the glycosyl bond, and the furanose ring assumes a 3'-exo pucker, which is different from that found in the complex with RNase T1. In the catalytic site of the RNase F1 complex with 2'GMP, one water molecule was observed, which bridges the phosphate oxygen atoms of 2'GMP and the side-chains of the catalytically important residues, His92 and Arg77, through hydrogen bonds. A water molecule occupying the same position was found in the RNase F1 free form. The significance of this water molecule in the hydrolytic reaction is discussed.
核糖核酸酶F1是一种来自串珠镰刀菌的鸟嘌呤特异性核糖核酸酶，在不存在抑制剂和存在2'-鸟苷酸（2'GMP）的情况下，以两种不同形式结晶。核糖核酸酶F1游离形式的晶体结构通过分子置换法解析，使用核糖核酸酶T1与2'GMP复合物的坐标，并使用扩展至1.3埃分辨率的数据精修至最终R因子为18.7%。对于核糖核酸酶F1与2'GMP复合物的晶体结构，分子置换法的解是基于核糖核酸酶F1游离形式的坐标获得的，并使用高达2埃分辨率的数据精修至最终R因子为16.8%。核糖核酸酶F1游离形式和与2'GMP复合物的两种晶体结构彼此非常相似，所有Cα原子的均方根位移（r.m.s.d.）值为0.43埃。两种结构之间主要差异与2'GMP在Tyr42和Glu46之间环中的底物识别位点的结合有关。核糖核酸酶F1和核糖核酸酶T1之间的结构比较显示所有Cα原子之间有显著相似性，均方根位移值为1.4埃证明了这一点。这两种酶中32至38位残基的环在构象和氢键模式上都有显著差异。催化活性残基His92的侧链相对于核糖核酸酶T1游离形式和核糖核酸酶T1与2'GMP复合物中的相应位置，在核糖核酸酶F1中从催化位点分别偏移了1.3埃和0.85埃。在核糖核酸酶F1复合物中，2'GMP的鸟嘌呤碱基围绕糖苷键呈顺式构象，呋喃糖环呈3'-外向型折叠，这与在与核糖核酸酶T1复合物中发现的不同。在核糖核酸酶F1与2'GMP复合物的催化位点观察到一个水分子，它通过氢键连接2'GMP的磷酸氧原子与催化重要残基His92和Arg77的侧链。在核糖核酸酶F1游离形式中发现了占据相同位置 的一个水分子。讨论了这个水分子在水解反应中的意义。
