STRUCTURAL CHARACTERIZATION OF REACTIVE OXYGEN DEFENCE ENZYMES: THE ENDONUCLEASE IV AND Y34F MUTANT MN SUPEROXIDE DISMUTASE. Yue Guan¹, Katrina Forest¹, Gloria Borgstahl¹, Michael Hickey¹, Richard Cunningham², John A. Tainer¹, ¹The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037, ²SUNY at Albany, Albany, NY 12222

The DNA repair enzyme endonuclease IV (endoIV), which is induced by superoxide, catalyzes the cleavage of DNA at apurinic/apyrimidinic (abasic or AP) sites resulting from reactive oxygen damage. It is critical for the survival of pathogens in the presence of host superoxide-mediated defenses. To understand the mechanism of damaged DNA detection and cleavage, we are solving structure of endoIV from E. coli and Mycobacterium leprae. These endoIV enzymes were crystallized in triclinic and monoclinic crystal forms. A 2.4-Å resolution native data set from the monoclinic crystal form (space group P2₁ with the unit cell dimensions of a=49Å, b=60Å, and c=5lÅ) has been collected at -180deg.C with flash cooling cryogenic device using MAR image plate area detector at SIEMENS rotating anode generator. We are now scaning crystals soaked in heavy atom compounds in order to find isomorphous derivatives to complete this new structure determination and current results on these structures will be presented.

Manganese superoxide dismutase (MnSOD) protects mitochondria against superoxide-mediated oxidative damage. MnSOD has usually high stability and fast catalysis. The structure of the native protein was previously solved at 2.2-Å resolution and Tyr 34 was proposed to serve as a proton carrier during the catalysis. To address the role of Tyr 34, the crystal structure of Y34F mutant MnSOD has been solved in two different crystal forms (P6₁22 and P2₁2_l2) using the molecular replacement method in the AmoRe program package. The structure for the hexagonal crystal form was refined to 1.9-Å resolution with the R-factor of 19% using the diffraction data collected at the UCSD Research Resource for Protein Crystallography. The orthorhombic form structure was refined to 2.0-Å resolution. Similar to the wild-type MnSOD, the crystal structure of the mutant is a homotetramer with Phe34 located in the active site. Each subunit is composed of the N-terminal helical hairpin domain and the C-terminal [[alpha]]/[[beta]] domains. Both domains contribute ligands to the catalytic manganese site. Current structural implications for MnSOD stability and activity will be presented