Investigation and application of hydrocarbon-degrading enzymes using cryo-electron microscopy and directed evolution

使用冷冻电子显微镜和定向进化研究和应用碳氢化合物降解酶

• 批准号: 10426459
• 负责人: Mary Catherine Andorfer
• 金额: $ 9.96万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10426459
• 关键词: Alkenes; Anaerobic Bacteria; Biochemical; Biological; Bioremediations; Chemicals; Chemistry; Corrosion; Coupled; Cryoelectron Microscopy; Development; Directed Molecular Evolution; Elements; Engineering; Environment; Enzymes; Equipment; Evolution; Faculty; Familiarity; Fumarates; Health; Heart Diseases; Human; Hydrocarbons; Hydrogen Bonding; In Vitro; Infrastructure; Institution; Investigation; Job Application; Kidney Diseases; Knowledge; Liver diseases; Manuscripts; Methods; Microbe; Microbiology; Molecular; Molecular Conformation; Nature; Oils; Organism; Oxygen; Petroleum; Phase; Pollution; Positioning Attribute; Postdoctoral Fellow; Process; Protein Engineering; Reaction; Research; Route; Ships; Site; Structure; Substrate Specificity; Succinates; Techniques; Technology; Training; Work; biophysical techniques; career; catalyst; cofactor; combat; design; fascinate; inhibitor; insight; interest; non-Native; novel; outreach; pollutant; remediation; structural biology; success; tool

PROJECT SUMMARY Glycyl radical enzymes (GREs) are a growing superfamily that catalyzes an impressive array of chemical transformations critical to both human health and the environment. GREs share a common glycyl radical cofactor which allows them to perform challenging, otherwise inaccessible chemistry; however, this simple yet effective cofactor is extremely oxygen sensitive. Because of the anaerobic nature of these catalysts, they are prevalent within oxygen-free environments such as the human gut, marine seeps, and crude-oil containing environments. GREs have been implicated in liver, heart, and kidney diseases and could prove uniquely effective as bioremediation tools and targets for biodeterioration inhibition; however, most GREs remain uncharacterized. Of particular interest is a class of GRE known as X-succinate synthases (XSSs), which are prevalent in hydrocarbon-degrading anaerobes. XSSs catalyze the hydroalkylation of fumarate, in which new C–C bonds are forged between fumarate and unactivated hydrocarbon substrates. This initial hydrocarbon-activation step allows for hydrocarbons to be further metabolized by these anaerobes. Through this mechanism, XSS-containing organisms are able to degrade hydrocarbon pollutants in even the most recalcitrant regions for environmental remediation. On the other hand, organisms with these enzymes also significantly contribute to microbiologically influenced corrosion. Beyond their potential environmental significance, XSS enzymes enable challenging chemistry and could serve as an important addition to the current C–H functionalization toolkit. The work described here will illuminate key missing mechanistic elements of XSSs and GREs more broadly, characterize new hydroalkylation enzymes, and explore GRE use in biocatalysis. Here, I aim to use cutting-edge cryo-electron microscopy (cryo-EM) tools and equipment to capture never-before-seen conformations of GREs as well as novel structures of XSS enzymes. Additionally, I aim to develop methods of installing the glycyl radical cofactor in vitro, a feat which has not yet been accomplished for any XSS enzyme to date. In vitro installation will allow us to probe details of hydroalkylation and activation mechanism that have been severely lacking for this class. Lastly, I will use directed evolution to engineer XSSs as selective hydroalkylation catalysts. Collectively, this work will provide insight into the ways in which Nature uses enzymes to achieve remarkable chemistry and will allow us to begin to harness the powerful radical chemistry Nature has to offer. I will complete the K99 phases of Aims 1 (develop a cryo-EM pipeline for XSSs using BSS) and 2 (determine conditions for in vitro activation of XSSs) during my postdoc in the Drennan lab at MIT. The R00 phases of Aims 1 (structural characterization of an alkyl- SS) and 2 (directed evolution of XSSs) will take place during my independent career. During the K99 phase, I will also develop other proposals for job applications, apply for faculty positions at research-intensive institutions, and continue my professional development through presentations, submission of manuscripts, and outreach activities.

项目摘要 甘氨酰自由基酶（GRES）是一个不断发展的超家族，催化一系列令人印象深刻的化学反应。 这些变化对人类健康和环境都至关重要。GRES共享一个共同的甘氨酰自由基辅因子 这使他们能够进行具有挑战性的，否则无法实现的化学;然而，这种简单而有效的 辅因子对氧非常敏感。由于这些催化剂的厌氧性质， 在无氧环境中，例如人类肠道、海洋渗漏和含原油的环境。 GRES与肝脏、心脏和肾脏疾病有关，并且可以证明其独特的有效性， 生物修复工具和生物退化抑制目标;然而，大多数绿色环境仍然没有特征。的 特别令人感兴趣的是一类称为X-琥珀酸脱氢酶（XSS）的GRE，其在哺乳动物中普遍存在。 烃降解厌氧菌。XSS催化富马酸酯的加氢烷基化，其中新的C-C键被 在富马酸盐和未活化的烃基质之间锻造。该初始烃活化步骤允许 以便这些厌氧微生物进一步代谢碳氢化合物。通过这种机制， 即使在环境污染最严重的地区，生物也能降解碳氢化合物污染物。 补救措施另一方面，具有这些酶的生物体也显著有助于微生物学上的 影响腐蚀。除了其潜在的环境意义外，XSS酶还可以实现具有挑战性的 化学，并可以作为一个重要的补充，目前的C-H官能化工具包。工作 这里所描述的将更广泛地阐明XSS和GRE的关键缺失机制元素， 新的加氢烷基化酶，并探索GRE在生物催化中的应用。在这里，我的目标是使用尖端的低温电子 显微镜（cryo-EM）工具和设备，以捕获以前从未见过的GR构象，以及 XSS酶的新结构。此外，我的目标是开发安装甘氨酰自由基辅因子的方法， 这是迄今为止任何XSS酶都没有完成的壮举。体外安装将允许 我们将探讨本课程严重缺乏的加氢烷基化和活化机制的细节。 最后，我将使用定向进化来设计XSS作为选择性加氢烷基化催化剂。总的来说，这项工作 将提供洞察自然界使用酶来实现显着化学的方式，并将允许 我们开始利用大自然提供的强大的自由基化学。我将完成目标的K99阶段 1（使用BSS开发XSS的冷冻EM管道）和2（确定XSS体外激活的条件） 我在麻省理工学院德伦南实验室做博士后期间。目的1的R 00相（烷基-N-甲基-N-苯基-N-甲基-N- SS）和2（XSS的定向进化）将在我的独立职业生涯中发生。在K99阶段，我 还将制定其他求职建议，申请研究密集型机构的教职， 并通过演讲，提交手稿和外展来继续我的专业发展 活动