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.

项目总结