CAREER: Mechanistic studies of the spore photoproduct lyase

职业：孢子光产物裂合酶的机理研究

• 批准号: 1454184
• 负责人: Lei Li
• 金额: $ 65万
• 依托单位: Indiana University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1454184&HistoricalAwards=false
• 关键词: CAREER; Mechanistic; studies; spore; photoproduct

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.

两种形成内生孢子的细菌菌株（肉毒杆菌和炭疽芽孢杆菌）是 A 类生物恐怖主义制剂。它们的内生孢子对紫外线照射具有极强的抵抗力。这种极不寻常的抗紫外线能力归因于一种称为孢子光产物（SP）的特殊DNA光损伤的形成，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允许选择性靶向SP修复过程，通过紫外线照射杀死孢子形成细菌，改善人类健康和国家安全。这一追求还将使研究生、本科生和高中生能够获得 DNA 光化学和机械酶学方面的专门培训。此外，通过建立胡西尔高中化学奥林匹克训练营，女性和代表性不足的少数族裔高中生将成为加强化学教育的目标。通过该奖项，化学系的生命过程化学项目正在资助印第安纳大学的李蕾博士-普渡大学印第安纳波利斯分校在孢子光产物裂合酶 (SPL) 中建立了一种新型自由基转移途径，并了解其在促进 S-腺苷甲硫氨酸 (SAM) 辅因子再生中的作用每个催化循环结束。非天然氨基酸将用于帮助表征该途径中假定的自由基种类。将采用荧光核苷酸类似物和氢/氘交换质谱来探测SPL催化过程中各自的DNA和蛋白质构象变化。拟议的研究将提供急需的信息，以揭示 SPL 中 SAM 再生的要求。由于 SAM 再生在所谓的自由基 SAM 超家族（有 56,000 个成员）中的许多其他酶中都有体现，因此所提出的研究对于我们不仅了解 SPL，而且了解该超家族中的其他酶都具有重要意义。

项目成果

Reversible Hydrolysis Reaction with the Spore Photoproduct under Alkaline Conditions

碱性条件下孢子光产物的可逆水解反应

• DOI: 10.1021/acs.joc.6b01846
• 发表时间: 2016
• 期刊: The Journal of Organic Chemistry
• 作者: Adhikari, Surya;Lin, Gengjie;Li, Lei
• 通讯作者: Li, Lei

EPR Study of UV-Irradiated Thymidine Microcrystals Supports Radical Intermediates in Spore Photoproduct Formation

紫外线照射胸苷微晶的 EPR 研究支持孢子光产物形成中的自由基中间体

• DOI: 10.1021/acs.jpcb.6b06587
• 发表时间: 2016
• 期刊: The Journal of Physical Chemistry B
• 作者: Hayes, Ellen C.;Jian, Yajun;Li, Lei;Stoll, Stefan
• 通讯作者: Stoll, Stefan