Abstract 1607 Characterization of a glycyl radical enzyme activating enzyme (GRE-AE) that equips anaerobes to functionalize inert hydrocarbons

Glycyl radical enzyme activating-enzymes (GRE-AEs) are members of the radical S-adenosyl-L-methionine (rSAM) superfamily and activate their cognate glycyl radical enzymes (GREs) by post-translational installation of the radical cofactor. Upon activation, GREs perform chemically challenging transformations that are inaccessible without the use of a radical cofactor. 4-isopropylbenzylsuccinate synthase (IBSS) is a GRE that activates inert hydrocarbons by C-H bond functionalization. First, IBSS activating-enzyme (IbsAE) installs a glycyl radical cofactor on IBSS, which then allows IBSS to catalyze the addition of p-cymene to fumarate in a hydroalkylation reaction to form 4-isopropylbenzylsuccinate. Of the thousands of bioinformatically identified GRE-AEs, only one activase–pyruvate formate-lyase activating enzyme (PFL-AE)–has been structurally characterized. Comparatively, PFL-AE is markedly different, as it lacks an auxiliary ferredoxin (Fdx) domain that is present in most other GRE-AEs, IbsAE included. In IbsAE, this Fdx domain contains eight conserved cysteines that may coordinate up to two [4Fe-4S] clusters. Fdx domains often play a role in electron transfer, yet the role of the Fdx domain in the GRE-AEs is unknown. Therefore, structurally characterizing a GRE-AE that contains this motif could provide insight on the function of the Fdx domain in the GRE-AE. To obtain a sample of IbsAE amenable to crystallization, we have made variants of the protein with N-terminal truncations and mutation of the cysteines that may coordinate [Fe-S] clusters. Anaerobic screening of these variants has yielded initial crystallization conditions. Our goal here is to solve a high-resolution crystal structure that may expand our understanding of GRE-AEs and the activation of GREs. Together with their GRE counterparts, GRE-AEs have potential applications in bioremediation, where they could be utilized to break down components of oil spills; in biotechnology, where they could be used to produce value-added chemical products and in biocatalysis, where they could be used to selectively set consecutive stereocenters on organic molecules. The Drennan Lab is funded by the Howard Hughes Medical Institute and the National Institute of Health.