Structural, thermodynamic, and mutational analyses of a psychrotrophic RNase HI
Structural, thermodynamic, and mutational analyses of a psychrotrophic RNase HI
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DOI：
10.1021/bi7001423
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发表时间：
2007-06-26
期刊：
影响因子：
2.9
通讯作者：
Kanaya, Shigenori
中科院分区：
文献类型：
--
作者：
Tadokoro, Takashi;You, Dong-Ju;Kanaya, Shigenori
来自嗜冷细菌Shewanella oneidensis MR-1的核糖核酸酶（RNase）HI在大肠杆菌中过量产生、纯化并进行结构和生化表征。MR-1 RNase HI的氨基酸序列与E. coli RNase HI。在2.0 A分辨率下测定的MR-1 RNase HI的晶体结构与E. coli RNase HI，但MR-1 RNase HI的分子内离子对数目和极性表面积比E. coli RNase HI。MR-1 RNase HI的酶学性质与E. coli RNase HI。但MR-1 RNase HI的稳定性远低于E. coli RNase HI。MR-1 RNase HI的热失活稳定性低于E. coliRNaseHI中。通过监测220 nm处的CD值，对MR-1 RNase HI的构象稳定性进行了分析。MR-1 RNase HI的稳定性不如E. coli RNase HI在22.4 ℃的T-m和12.5 kJ/mol的Delta G（H2O）中的降解。MR-1 RNase HI的热力学稳定性曲线与E. coli RNase HI。定点突变研究表明，分子内离子对数目的差异部分地解释了MR-1和E. coli RNases HI.
Ribonuclease (RNase) HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 was overproduced in Escherichia coli, purified, and structurally and biochemically characterized. The amino acid sequence of MR-1 RNase HI is 67% identical to that of E. coli RNase HI. The crystal structure of MR-1 RNase HI determined at 2.0 A resolution was highly similar to that of E. coli RNase HI, except that the number of intramolecular ion pairs and the fraction of polar surface area of MR-1 RNase HI were reduced compared to those of E. coli RNase HI. The enzymatic properties of MR-1 RNase HI were similar to those of E. coli RNase HI. However, MR-1 RNase HI was much less stable than E. coli RNase HI. The stability of MR-1 RNase HI against heat inactivation was lower than that of E. coli RNase HI by 19 degrees C. The conformational stability of MR-1 RNase HI was thermodynamically analyzed by monitoring the CD values at 220 nm. MR-1 RNase HI was less stable than E. coli RNase HI by 22.4 degrees C in T-m and 12.5 kJ/mol in Delta G(H2O). The thermodynamic stability curve of MR-1 RNase HI was characterized by a downward shift and increased curvature, which results in an increased Delta C-p value, compared to that of E. coli RNase HI. Site-directed mutagenesis studies suggest that the difference in the number of intramolecular ion pairs partly accounts for the difference in stability between MR-1 and E. coli RNases HI.