Ishikawa K, Okumura M, Katayanagi K, Kimura S, Kanaya S, Nakamura H, Morikawa K
Protein Engineering Research Institute, Osaka, Japan.
J Mol Biol. 1993 Mar 20;230(2):529-42. doi: 10.1006/jmbi.1993.1169.
The crystal structure of Thermus thermophilus RNase H was determined at 2.8 A resolution. The structure was solved by the molecular replacement method, based on the accurately refined structure of Escherichia coli RNase HI, which shows 52% amino acid sequence identity. Crystallographic refinement led to an R-factor of 0.205, with a 0.019 A root-mean-square deviation from ideal bond lengths and 0.048 A from ideal bond angle distances. Structural comparison shows a striking similarity in the overall folding of the thermophilic and mesophilic enzymes. The root-mean-square displacement is 0.95 A between equivalent alpha-carbon atoms from all elements of secondary structure (five alpha-helices and five beta-strands). However, some notable differences, which account for the enhanced thermostability of T. thermophilus RNase H, are observed in loop structures and side-chain conformations. The substitution of Gly for the left-handed helical residue (Lys95) in the E. coli enzyme is proposed to substantially enhance the thermostability, due to the release of steric hindrance caused by the beta-carbon atom. Furthermore, it is likely that the expansion of an aromatic cluster, arising from the replacement of Ile78 in the mesophilic enzyme by Phe, and the increased number of salt-bridges additively contribute to the stability.
嗜热栖热菌核糖核酸酶H的晶体结构在2.8埃分辨率下被测定。该结构通过分子置换法解析，基于大肠杆菌核糖核酸酶HI精确优化的结构，二者氨基酸序列一致性为52%。晶体学优化得到的R因子为0.205，与理想键长的均方根偏差为0.019埃，与理想键角的偏差为0.048埃。结构比较显示嗜热酶和嗜温酶在整体折叠上有显著相似性。来自所有二级结构元件（五个α螺旋和五个β链）的等效α碳原子之间的均方根位移为0.95埃。然而，在环结构和侧链构象中观察到一些显著差异，这些差异解释了嗜热栖热菌核糖核酸酶H增强的热稳定性。有人提出，大肠杆菌酶中左手螺旋残基（Lys95）被甘氨酸取代，由于β碳原子引起的空间位阻的释放，可大幅提高热稳定性。此外，嗜温酶中Ile78被苯丙氨酸取代导致的芳香簇扩展以及盐桥数量的增加可能共同有助于稳定性的提高。
