CAREER: Mechanistic studies of the spore photoproduct lyase

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Two endospore-forming bacterial strains (Clostridium botulinum and Bacillus anthracis) are Category A bioterrorism agents. Their endospores are extremely resistant to UV irradiation. This highly unusual UV resistance is ascribed to the formation of a special DNA photo-lesion called the spore photoproduct (SP), and the efficient repair of SP by the enzyme spore photoproduct lyase (SPL). The pursuit of the reaction mechanism of SPL may allow the selective targeting of the SP repair process to kill spore-forming bacteria via UV irradiation, improving human health and national security. This pursuit will also permit graduate, undergraduate, and high school students to acquire specialized training in DNA photochemistry and mechanistic enzymology. Additionally, female and underrepresented minority high school students will be targeted for enhanced chemistry education by establishing the Hoosier High School Chemistry Olympiad Training Camp.With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Lei Li from Indiana University-Purdue University Indianapolis to establish the presence of a novel radical transfer pathway in spore photoproduct lyase (SPL) and understand its role in facilitating the regeneration of the S-adenosylmethionine (SAM) cofactor at the end of each catalytic cycle. Unnatural amino acids will be utilized to help characterize the putative radical species along the pathway. Fluorescent nucleotide analogs and hydrogen/deuterium exchange mass spectrometry will be adopted to probe the respective DNA and protein conformational changes during SPL catalysis. The proposed studies will provide the much-needed information to reveal the requirements enabling SAM regeneration in SPL. As SAM regeneration is indicated in many other enzymes in the so-called radical SAM superfamily ( 56,000 members), the proposed studies are of great significance to our understanding of not only SPL, but also the other enzymes in the superfamily.