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

Glycyl radical enzymes (GREs) are a growing superfamily that catalyzes an impressive array of chemical transformations critical to human health. 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 petroleum 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 can 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 in crude-oil facilities. Beyond this 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. In vitro installation will allow us to probe details of hydroalkylation and activation mechanisms 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 have completed the K99 phases of Aims 1 (develop a cryo-EM pipeline for XSSs) and 2 (determine conditions for in vitro activation of XSSs) during my postdoc in the Drennan lab at MIT. I will work to complete the R00 phases of Aims 1 (Probe the structure and mechanism of XSSs) and 2 (Develop XSSs as selective hydroalkylation catalysts).