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Changing the Metal Binding Specificity of Superoxide Dismutase from Thermus thermophilus HB-27 by a Single Mutation

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Abstract

Metal binding of superoxide dismutase from Thermus thermophilus HB27 was analyzed by comparing the related structures and sequences from different origins. Mutants (Ile166Leu, Asp167Glu, and Ile166Leu-Asp167Glu) were prepared and characterized. The mutants Asp167Glu and Ile166Leu-Asp167Glu changed their binding specificities from manganese to iron, which were manifested by the differences in color of the enzyme solutions and by flame atomic absorption analysis. Specific activities of the three mutants were 112, 52, and 62% of that of the wild-type enzyme, respectively. Asp167Glu and Ile166Leu-Asp167Glu only retained 6.8 and 6.1%, respectively, of the original activities after dialysis against 1 mM EDTA. Tryptophan fluorescence measurement and native gel electrophoresis implied that the three mutants could fold into a less condensed structure. Their folding and changes in the ion binding sites of the modeled structures might be the reason for their low affinities to metal ions. These findings increased our understanding of metal binding specificity of superoxide dismutase.

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Acknowledgments

We will give our sincere thanks to Dr. Hu Zhu (China University of Petroleum, East China) for careful reading of the manuscript.

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Authors and Affiliations

  1. School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, 214122, People’s Republic of China

    Tianwen Wang

  2. Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, People’s Republic of China

    Tianwen Wang, Aidong Qiu & Haimeng Zhou

  3. Yangtze Delta Region Institute of Tsinghua University, Jiaxing, Zhejiang, 314100, People’s Republic of China

    Fanguo Meng & Haimeng Zhou

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  1. Tianwen Wang
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Correspondence to Tianwen Wang.

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Wang, T., Qiu, A., Meng, F. et al. Changing the Metal Binding Specificity of Superoxide Dismutase from Thermus thermophilus HB-27 by a Single Mutation. Mol Biotechnol 42, 146–153 (2009). https://doi.org/10.1007/s12033-009-9149-9

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  • DOI: https://doi.org/10.1007/s12033-009-9149-9

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