Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity

Anaerobic degradation of the environmental pollutant toluene is initiated by the glycyl radical enzyme benzylsuccinate synthase (BSS), which catalyzes the radical addition of toluene to fumarate, forming benzylsuccinate. We have determined crystal structures of the catalytic alpha-subunit of BSS with its accessory subunits beta and gamma, which both bind a [4Fe-4S] cluster and are essential for BSS activity in vivo. We find that BSS alpha has the common glycyl radical enzyme fold, a 10-stranded beta/alpha-barrel that surrounds the glycyl radical cofactor and active site. Both accessory subunits beta and gamma display folds related to high potential iron-sulfur proteins but differ substantially from each other in how they interact with the alpha-subunit. BSS gamma binds distally to the active site, burying a hydrophobic region of BSS alpha, whereas BSS beta binds to a hydrophilic surface of BSS alpha that is proximal to the active site. To further investigate the function of BSS beta, we determined the structure of a BSS alpha gamma. complex. Remarkably, we find that the barrel partially opens, allowing the C-terminal region of BSS alpha that houses the glycyl radical to shift within the barrel toward an exit pathway. The structural changes that we observe in the BSS alpha gamma complex center around the crucial glycyl radical domain, thus suggesting a role for BSS beta in modulating the conformational dynamics required for enzyme activity. Accompanying proteolysis experiments support these structural observations.