title: Changing the metal binding specificity of superoxide dismutase from Thermus thermophilus HB-27 by a single mutation.
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.
authors.affiliation: Jiangnan University, Wuxi, Jiangsu, People's Republic of China. theobald.wang@yahoo.com.cn
authors.forename: Tianwen
authors.initials: T
authors.lastname: Wang
authors.forename: Aidong
authors.initials: A
authors.lastname: Qiu
authors.forename: Fanguo
authors.initials: F
authors.lastname: Meng
authors.forename: Haimeng
authors.initials: H
authors.lastname: Zhou
mesh_terms: D001665:Binding Sites; D004789:Enzyme Activation; D007501:Iron / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016963:Thermus thermophilus / Q000235:genetics* / Q000378:metabolism*
chemical_list: D008345:Manganese; D007501:Iron; D013482:Superoxide Dismutase