The metalloenzyme Cu,Zn superoxide dismutase (SOD) is a master eukaryotic regulator of reactive oxygen species within cells by dismutating superoxide anions into peroxide and oxygen. Biochemically, SOD is remarkable for its unusually great subunit and dimer stability, faster than diffusion enzyme-substrate recognition coupled to exquisite specificity, and efficient catalysis requiring alternate superoxide oxidation and reduction. Biologically, SOD is important for decreasing aging and increasing lifespan by reducing oxidative stress from inflammation and injury. Medically, human SOD or HSOD is notable for the recently proven role of SOD mutations in causing the fatal degenerative disease of motor neurons termed amyotrophic lateral sclerosis (ALS) or Lou Gherig's disease. Our structures of human, bovine, yeast, and Photobacter SODs provide a basis for the proposed studies that focus on the structural metallobiochemistry of ALS HSOD mutants. High resolution structure determinations of HSOD mutants will be integrated with coupled computational, mutational, and biochemical analyses including assays of stability, assembly, metal binding, and catalytic activity. Due to the uncertainties in mutant design and crystallization, a pyramid strategy is proposed starting with the large base of SOD ALS mutants plus mutants designed to probe HSOD folding, stability, assembly, and activity. After expression and preliminary characterization, the focus will be on mutants that prove most informative biochemically, and then to the subset of key mutants that provide crystals suitable for high resolution HSOD structures. Biochemical and biological results on our mutant SODs obtained by our collaborators will complement research on HSOD structural metallobiochemistry at Scripps. The molecular basis for possible toxic properties of ALS SOD mutants including peroxidation and nitration activities will be defined in terms of active site architecture. The combination of ALS variants with other designed mutants (altered in HSOD folding, stability, assembly, metal binding, substrate recognition, specificity, active channel architecture, or catalysis) will provide enzymes to be used in transgenic mice and Drosophila systems to test hypotheses regarding the molecular basis for ALS. HSOD mutations that alter HSOD assembly, cell surface binding, and half-life will provide protein lead compounds for possible eventual therapeutics. Overall, the proposed research will improve fundamental understanding of SOD metallobiochemistry, structure, and function at the atomic level and contribute to understanding how HSOD variants can cause ALS.
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