Remarkable Stabilization of a Psychrotrophic RNase HI by a Combination of Thermostabilizing Mutations Identified by the Suppressor Mutation Method

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• 作者：Takashi Tadokoro ; Kyoko Matsushita ; Yumi Abe ; Muhammad Saifur Rohman ; Yuichi Koga ; Kazufumi Takano ; Shigenori Kanaya
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：August 5, 2008
• 年：2008
• 卷：47
• 期：31
• 页码：8040-8047
• 全文大小：235K
• 年卷期：v.47,no.31(August 5, 2008)
• ISSN：1520-4995

文摘

Ribonuclease HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 (So-RNase HI) is much less stable than Escherichia coli RNase HI (Ec-RNase HI) by 22.4 °C in T_m and 12.5 kJ mol⁻¹ in ΔG(H₂O), despite their high degrees of structural and functional similarity. To examine whether the stability of So-RNase HI increases to a level similar to that of Ec-RNase HI via introduction of several mutations, the mutations that stabilize So-RNase HI were identified by the suppressor mutation method and combined. So-RNase HI and its variant with a C-terminal four-residue truncation (154-RNase HI) complemented the RNase H-dependent temperature-sensitive (ts) growth phenotype of E. coli strain MIC3001, while 153-RNase HI with a five-residue truncation could not. Analyses of the activity and stability of these truncated proteins suggest that 153-RNase HI is nonfunctional in vivo because of a great decrease in stability. Random mutagenesis of 153-RNase HI using error-prone PCR, followed by screening for the revertants, allowed us to identify six single suppressor mutations that make 153-RNase HI functional in vivo. Four of them markedly increased the stability of the wild-type protein by 3.6−6.7 °C in T_m and 1.7−5.2 kJ mol⁻¹ in ΔG(H₂O). The effects of these mutations were nearly additive, and combination of these mutations increased protein stability by 18.7 °C in T_m and 12.2 kJ mol⁻¹ in ΔG(H₂O). These results suggest that several residues are not optimal for the stability of So-RNase HI, and their replacement with other residues strikingly increases it to a level similar to that of the mesophilic counterpart.