Subunit Structure of Benzylsuccinate Synthase

Abstract

Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits α, β and γ that in Thauera Aromatica T1 strain are encoded by the tutD, tutG and tutF genes respectively. The large α-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small β- and γ-subunits has remained unclear. We have over-expressed all three subunits of benzylsuccinate synthase in E. coli, both individually and in combination. Co-expression of the γ-subunit (but not the β-subunit) is essential for efficient expression of the α-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an α2β2γ2 hexamer by nickel-affinity chromatography through a ‘His6’ affinity tag engineered onto the C-terminus of the α-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the β-subunit and the other with the γ-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly a mutant γ-subunit lacking the [4Fe-4S] cluster is unable to stabilize the α-subunit when the proteins are co-expressed.

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

“…Previous studies on BSS have shown that expression and folding of BSSα depends on the coexpression of BSSγ but not BSSβ (16). The structure of the complex reveals a mostly hydrophobic binding site for BSSγ (Fig.…”

“…The clusters of both BSSβ and BSSγ do appear to play a structural role in that their removal leads to dissociation of the subunits from BSSα (16), which in turn leaves BSSα prone to unfolding and clearance by cellular degradation machinery in vivo (16) and sensitive to proteases in vitro. In terms of whether these clusters also play a redox role in addition to the above structural role, we find that the overall fold of both subunits is derived from a HiPIP, a family of [4Fe-4S] cluster proteins known for their high redox potentials (100-450 mV) and for their ability to stabilize clusters in the +3 oxidation state (20,27).…”

“…C-terminally His-tagged BSSα was coexpressed with either BSSγ alone or BSSγ and BSSβ and purified as previously described (16). Crystals of the BSSαβγ complex were grown by vapor diffusion.…”

“…heterohexamer can be isolated from cultured Thauera aromatica strain T1 grown on toluene (15) or expressed recombinantly in Escherichia coli (16). The 98-kDa α-subunit contains the catalytic machinery and forms a homodimeric complex, a common feature of GREs.…”

“…The 98-kDa α-subunit contains the catalytic machinery and forms a homodimeric complex, a common feature of GREs. BSSβ and -γ are much smaller, 9 and 7 kDa, respectively, and have been investigated on their own (16) and in complex with the α-subunit (16,17). Electron paramagnetic resonance, Mössbauer, and UV-visible spectroscopy from these studies indicate that BSSβ and BSSγ each contain a [4Fe-4S] cluster ligated by four conserved cysteines in these proteins.…”