CAREER: Mechanistic studies of the spore photoproduct lyase

两种内孢子形成细菌菌株（肉毒杆菌和炭疽芽孢杆菌）是A类生物恐怖制剂。它们的内生孢子对紫外线照射有极强的抵抗力。这种极不寻常的抗紫外线能力归因于形成一种特殊的DNA光损伤，称为孢子光产物（SP），以及孢子光产物裂解酶（SPL）对SP的有效修复。对SPL反应机制的探索，可能使SPL修复过程选择性靶向，通过紫外线照射杀死孢子形成细菌，从而改善人类健康和国家安全。这种追求也将允许研究生，本科生和高中生获得DNA光化学和机械酶学的专门训练。此外，通过建立印第安纳州高中化学奥林匹克训练营，女性和少数族裔高中学生将成为加强化学教育的目标。有了这个奖项，化学学部的生命过程化学项目资助了来自印第安纳大学-普度大学印第安纳波利斯分校的李雷博士，以建立孢子光产物裂解酶（SPL）中新型自由基转移途径的存在，并了解其在促进s -腺苷甲硫氨酸（SAM）辅助因子在每个催化循环结束时的再生中的作用。将利用非天然氨基酸来帮助表征沿该途径推定的自由基物种。将采用荧光核苷酸类似物和氢/氘交换质谱法探测SPL催化过程中各自的DNA和蛋白质构象变化。建议进行的研究将提供急需的资料，以揭示在森林公园内进行SAM再生所需的条件。由于SAM再生在所谓的自由基SAM超家族（56,000个成员）中的许多其他酶中都有表现，因此所提出的研究不仅对我们了解SPL，而且对超家族中的其他酶具有重要意义。

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.