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

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

Abstract

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)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

RENGIFO, EMERITA MENDOZA; FONTOLAN, LAUREANA STELMASTCHUK BENASSI; FERREIRA-JUNIOR, JOSE RIBAMAR; BLEICHER, LUCAS; PENNER-HAHN, JAMES; GARRATT, RICHARD CHARLES. Unexpected plasticity of the quaternary structure of iron-manganese superoxide dismutases. Journal of Structural Biology, v. 214, n. 2, p. 11-pg., 2022-04-11. (14/01855-2)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

RENGIFO, Emérita Mendoza. Exploring the selectivity of metal ions in the active site of the enzyme superoxide dismutase (SOD) using site-directed mutagenesis. 2016. Master's Dissertation - Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT) São Carlos.