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: 10868997
• 负责人: Mary Catherine Andorfer
• 金额: $ 24.9万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 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 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10868997
• 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.

项目概要 甘氨酰自由基酶 (GRE) 是一个不断发展的超家族，可催化​​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化具有共同的甘氨酰自由基辅助因子。 这使得他们能够进行具有挑战性的、原本难以实现的化学反应；然而，这种简单而有效的化学反应； 由于这些催化剂的厌氧性质，辅助因子对氧极其敏感。 在无氧环境中，例如人类肠道、海羊和含有原油的环境中。 GRE 与肝脏、心脏和肾脏疾病有关，并且可能被证明具有独特的功效： 生物修复工具和抑制生物恶化的目标；然而，大多数 GRE 仍未得到表征。 特别令人感兴趣的是一类被称为 X-琥珀酸合酶 (XSS) 的 GRE，它普遍存在于 XSS 催化富马酸的加氢烷基化，其中产生新的 C-C 键。 在富马酸盐和未活化的碳氢化合物基质之间锻造，该初始碳氢化合物活化步骤允许。 通过这种机制，碳氢化合物可以被这些厌氧菌进一步代谢。 即使在环境最恶劣的地区，生物体也能够降解碳氢化合物污染物 另一方面，具有这些酶的生物体也对微生物学有显着贡献。 除了潜在的环境意义外，XSS 酶还具有挑战性。 化学，可以作为当前 C-H 功能化工具包的重要补充。 这里描述的将更广泛地阐明 XSS 和 GRE 中缺失的关键机械元素，表征 新型加氢烷基化酶，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（冷冻电镜）工具和设备，用于捕获从未见过的 GRE 构象以及 此外，我的目标是开发安装甘氨酰自由基辅助因子的方法。 在体外，这是迄今为止任何 XSS 酶都尚未实现的壮举。 我们探索此类药物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。 将深入了解大自然如何利用酶来实现卓越的化学反应，并允许 我们将开始利用大自然提供的强大的自由基化学作用，我将完成目标的 K99 阶段。 1（使用 BSS 开发 XSS 的冷冻电镜管道）和 2（确定 XSS 体外激活的条件） 在麻省理工学院 Drennan 实验室做博士后期间，目标 1 的 R00 相（烷基-的结构表征） SS）和2（XSS的定向进化）将发生在我的独立职业生涯中，我的K99阶段。 还将制定其他工作申请提案，申请研究密集型机构的教职职位， 并通过演讲、提交手稿和外展继续我的专业发展 活动。