CAREER: Mechanistic studies of the spore photoproduct lyase

两株形成内孢子的细菌(肉毒杆菌和炭疽芽孢杆菌)是A类生物恐怖分子。它们的内孢子对紫外线具有极强的抵抗力。这种极不寻常的紫外线抗性归因于一种特殊的DNA光损伤的形成，称为孢子光产物(SP)，并通过酶孢子光产物裂解酶(SPL)有效地修复SP。对SPL反应机制的追求可能使SP修复过程成为选择性靶向，通过紫外线照射杀死芽胞形成菌，改善人类健康和国家安全。这一追求也将允许研究生、本科生和高中生获得DNA光化学和机械酶学方面的专门培训。此外，针对女生和代表性不足的少数族裔高中生，将通过建立Hoosier高中化学奥林匹克训练营来加强化学教育。获得这个奖项的是，化学系生命过程化学项目资助印第安纳大学-普渡大学印第安纳波利斯分校的雷丽博士建立孢子光产物裂解酶(SPL)中存在的一种新的自由基转移途径，并了解其在促进每个催化循环结束时S-腺苷蛋氨酸辅因子再生中的作用。非天然氨基酸将被用来帮助描述该途径中假定的自由基物种。将采用荧光核苷酸类似物和氢/氚交换质谱仪来探测SPL催化过程中DNA和蛋白质的构象变化。拟议的研究将提供急需的信息，以揭示在SPL中实现SAM再生的要求。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表明，所提出的研究不仅对我们理解SPL，而且对超家族中的其他酶都具有重要意义。

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.