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

项目总结 甘氨酰自由基酶(GREs)是一个正在成长的超家族，它催化着一系列令人印象深刻的化学物质 对人类健康和环境都至关重要的转变。GRE共有一个共同的甘氨酸基辅助因子 这使他们能够进行具有挑战性的、否则无法接触到的化学；然而，这一简单而有效的方法 辅因子对氧极为敏感。由于这些催化剂的厌氧性，它们普遍存在。 在无氧环境中，如人类肠道、海洋渗漏和含原油环境中。 GRES与肝脏、心脏和肾脏疾病有关，并可能被证明是唯一有效的 抑制生物退化的生物修复工具和目标；然而，大多数GRE仍然没有特征。的 特别令人感兴趣的是一类被称为X-琥珀酸合成酶(XSS)的GRE，它普遍存在于 降解碳氢化合物的厌氧菌。XSS催化富马酸氢烷基化反应，其中新的C-C键是 在富马酸和未活化的碳氢化合物底物之间锻造。这一初始的碳氢化合物活化步骤允许 碳氢化合物被这些厌氧菌进一步代谢。通过这种机制，包含XSS的 生物能够在环境污染最顽固的地区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对微生物有重大贡献。 影响腐蚀。除了其潜在的环境意义，XSS酶使具有挑战性的 化学，并可作为当前C-H官能化工具包的重要补充。这项工作 此处描述的内容将更广泛地说明XSS和GRE缺少的关键机械要素， 新的氢烷基化酶，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(冷冻-EM)工具和设备，用于捕捉前所未见的GRE构象以及 XSS酶的新结构。此外，我的目标是开发安装甘氨酰基辅助因子的方法 在体外，到目前为止还没有任何XSS酶完成这一壮举。体外安装将允许 美国将探索氢烷基化和活化机制的细节，而这一类严重缺乏这些细节。 最后，我将利用定向进化来设计XSS作为选择性氢烷基化催化剂。总体而言，这项工作 将提供对大自然如何使用酶来实现非凡化学的洞察，并将允许 我们要开始利用大自然提供的强大的激进化学。我将完成AIMS的K99阶段 1(使用BSS开发用于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瑞南实验室做博士后时。AIMS 1的R00相(烷基-1的结构表征 SS)和2(XSS的定向进化)将发生在我独立的职业生涯中。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师职位， 并通过演讲、提交手稿和外展来继续我的专业发展 活动。

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.