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.
