Subunit Structure of Benzylsuccinate Synthase

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 strain T1 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 overexpressed all three subunits of benzylsuccinate synthase in Escherichia coli, both individually and in combination. Coexpression 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. Un...