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Exploring the selectivity of metal ions in the active site of superoxide dismutase enzyme using site directed mutagenesis

Grant number: 14/01855-2
Support type:Scholarships in Brazil - Master
Effective date (Start): September 01, 2014
Effective date (End): August 31, 2016
Field of knowledge:Biological Sciences - Biophysics - Molecular Biophysics
Cooperation agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
Principal researcher:Richard Charles Garratt
Grantee:Emérita Mendoza Rengifo
Home Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil


Superoxide Dismutases (SODs) are metalloenzymes, used as a defense against oxidative damage caused by Reactive Oxygen Species (ROS) in aerobic organisms, through the conversion of superoxide anion into molecular oxygen (O2) and hydrogen peroxide (H2O2). Superoxide dismutases of Fe and Mn have sequences that are highly conserved, especially those residues that are around the active site (including all direct metal ligands), and their crystal structures superimpose well. However, with few exceptions, each enzyme is specific for only one of the two metals and the metal in the active site is changed (iron by manganese or vice versa) the result is a non- functional enzyme. This indicates that these proteins have high specificity for metal whose origin cannot be explained trivially. Bachega and colleagues made a statistical coupling analysis (SCA, the English Statistical Coupling Analysis) in the family of Fe/MnSODs where analysis showed that statistically coupled amino acids are clustered near the nucleus responsible for the formation of the metal centers. The most interesting was that the statistically coupled residues were different in the two groups of enzymes, so being able to distinguish specific enzymes for specific Fe by Mn. This result suggests that the residues identified in the analysis SCA may be responsible for fine-tuning of specificity metal. The objective of this research project is to identify the structural determinants of fine-tuning the specificity of metal MnSOD and FeSOD. Initially, we intend to select residues for site- directed mutagenesis MnSOD in the organism Trichoderma reesei (TrMnSOD). Then mutants will be expressed, purified and crystallized. The three-dimensional structure of the mutant will be resolved by X-ray diffraction and determined its enzymatic activity. The presence of metals and information about their neighborhood will be obtained by Electron Paramagnetic Resonance. Our hypothesis is that the SCA analysis indicates amino acids candidates for site-directed mutagenesis in order to design new SODs with intermediate binding characteristics of the Mn/Fe, and even the possibility of interconverting specificity. With this we aim to contribute to our understanding of the structural details of the fine-tuning of specificity. The proposal is basic research and is part of an overarching theme: the relationship between structure and function of proteins. (AU)

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