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: 10650407
• 负责人: Mary Catherine Andorfer
• 金额: $ 1.25万
• 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 2023-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10650407
• 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; Molecular; Molecular Conformation; Nature; Oils; Organism; Oxygen; Petroleum; Phase; Pollution; Positioning Attribute; Postdoctoral Fellow; Process; Protein Engineering; Reaction; Research; Route; Site; Structure; Substrate Specificity; Succinates; Techniques; Technology; Training; Work; biophysical techniques; career; catalyst; cofactor; combat; design; fascinate; forging; inhibitor; insight; interest; marine; 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 糖基自由基酶（GRES）是一种增长的超家族，催化了令人印象深刻的化学阵列 对人类健康和环境至关重要的转变。 GRE共享一个常见的糖基自由基辅助因子 这使他们能够执行挑战，否则难以接近的化学反应；但是，这个简单而有效 辅因子对氧气敏感。由于这些催化剂的厌氧性，它们很普遍 在无氧环境中，例如人类肠道，海洋看到和包含原油的环境。 肝脏，心脏和肾脏疾病中隐含了GRE，可以被证明是独特的 生物修复工具和生物临时抑制的目标；但是，大多数GRE仍然没有表征。的 特别有趣 碳化碳质厌氧菌。 XSS催化富马酸盐的氢烷基化，其中新的C – C键为 在富马酸盐和未活化的烃底物之间锻造。这个初始的烃激活步骤允许 这些厌氧菌将进一步代谢碳氢化合物。通过这种机制，含XSS 生物体甚至能够在最顽固的环境区域中降解烃污染物 修复。另一方面，使用这些酶的生物也显着有助于微生物学 影响腐蚀。 XSS酶超越其潜在的环境意义，使挑战者 化学，可以作为当前C – H功能化工具包的重要补充。工作 此处描述的将更广泛地阐明XSS和GRE的密钥缺失的机械元素，以表征 新的氢烷基化酶，并探索GRE在生物催化中的使用。在这里，我的目标是使用尖端的低温电子 显微镜（冷冻EM）工具和设备，以捕获GRE和GRE的构型之前 XSS酶的新结构。此外，我旨在开发安装糖基自由基辅助因子的方法 在体外，迄今为止，任何XSS酶都尚未实现这一壮举。体外安装将允许 我们探究了该类别缺乏水烷基化和激活机制的细节。 最后，我将使用定向进化来设计XSSS作为选择性水烷基化催化剂。总的来说，这项工作 将提供有关大自然使用酶实现非凡化学反应的方式的见解，并将允许 我们开始利用强大的激进化学性质必须提供的。我将完成目标的K99阶段 1（使用BSS开发XSS的冷冻管道）和2（确定XSS体外激活的条件） 在我在麻省理工学院的Drennan实验室的博士后。目标1的R00阶段（烷基的结构表征 SS）和2（XSSS的指示演变）将在我的独立职业生涯中发生。在K99阶段，我 还将为职位申请制定其他建议，申请研究密集型机构的教师职位， 并通过演讲，提交手稿和宣传来继续我的专业发展 活动。