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Article

Structural, Thermodynamic, and Mutational Analyses of a Psychrotrophic RNase HI^†^,^‡

Takashi Tadokoro,^§ Dong-Ju You,^§ Yumi Abe,^§ Hyongi Chon,^§ Hiroyoshi Matsumura,^@ Yuichi Koga,^§ Kazufumi Takano,^§^@ and Shigenori Kanaya*^§

Department of Material and Life Science and Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan, and CREST, JST, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

Biochemistry, 2007, 46 (25), pp 7460–7468

DOI: 10.1021/bi7001423

Publication Date (Web): May 31, 2007

†

This work was supported in part by a Grant-in-Aid for National Project on Protein Structural and Functional Analyses and by a Grant-in-Aid for Scientific Research on Priority Areas “Systems Genomics” from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by an Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.

‡

The Protein Data Bank entry for MR-1 RNase HI is 2E4L.

Department of Material and Life Science, Graduate School of Engineering, Osaka University.

Present address: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20892.

Department of Applied Chemistry, Graduate School of Engineering, Osaka University.

CREST.

To whom correspondence should be addressed. Telephone and fax: +81-6-6879-7938. E-mail: kanaya@mls.eng.osaka-u.ac.jp.

Abstract

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 Å 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 °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 °C in T_m and 12.5 kJ/mol in ΔG(H₂O). The thermodynamic stability curve of MR-1 RNase HI was characterized by a downward shift and increased curvature, which results in an increased ΔC_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.