title: Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity.
abstract: 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 α-subunit of BSS with its accessory subunits β and γ, which both bind a [4Fe-4S] cluster and are essential for BSS activity in vivo. We find that BSSα has the common glycyl radical enzyme fold, a 10-stranded β/α-barrel that surrounds the glycyl radical cofactor and active site. Both accessory subunits β and γ display folds related to high potential iron-sulfur proteins but differ substantially from each other in how they interact with the α-subunit. BSSγ binds distally to the active site, burying a hydrophobic region of BSSα, whereas BSSβ binds to a hydrophilic surface of BSSα that is proximal to the active site. To further investigate the function of BSSβ, we determined the structure of a BSSαγ complex. Remarkably, we find that the barrel partially opens, allowing the C-terminal region of BSSα that houses the glycyl radical to shift within the barrel toward an exit pathway. The structural changes that we observe in the BSSαγ complex center around the crucial glycyl radical domain, thus suggesting a role for BSSβ in modulating the conformational dynamics required for enzyme activity. Accompanying proteolysis experiments support these structural observations.
authors: Department of Chemistry.
authors: Michael A
authors: MA
authors: Funk
authors: Department of Chemistry, Boston University, Boston, MA 02215; and.
authors: Evan T
authors: ET
authors: Judd
authors: Departments of Chemistry and Biological Chemistry, University of Michigan, Ann Arbor, MI 48109.
authors: E Neil G
authors: EN
authors: Marsh
authors: Department of Chemistry, Boston University, Boston, MA 02215; and.
authors: Sean J
authors: SJ
authors: Elliott
authors: Department of Chemistry,Department of Biology, andHoward Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139; cdrennan@mit.edu.
authors: Catherine L
authors: CL
authors: Drennan
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D019755:Carbon-Carbon Lyases / Q000737:chemistry*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D005609:Free Radicals / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D020628:Thauera / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D005609:Free Radicals; D007506:Iron-Sulfur Proteins; D019755:Carbon-Carbon Lyases; C114354:benzylsuccinate synthase; D005998:Glycine
keywords: bioremediation; crystallography; iron–sulfur cluster; microbial metabolism; radical chemistry