Structural characterization of cobalamin-dependent radical S-adenosylmethionine methylases.

Cobalamin-dependent radical S-adenosylmethionine (SAM) methylases catalyze key steps in the biosynthesis of numerous biomolecules, including protein cofactors, antibiotics, herbicides, and other natural products, but have remained a relatively understudied subclass of radical SAM enzymes due to their inherent insolubility upon overproduction in Escherichia coli. These enzymes contain two cofactors: a [4Fe-4S] cluster that is ligated by three cysteine residues, and a cobalamin cofactor typically bound by residues in the N-terminal portion of the enzyme. Recent advances in the expression and purification of these enzymes in their active states and with both cofactors present has allowed for more detailed biochemical studies as well as structure determination by X-ray crystallography. Herein, we use KsTsrM and TokK to highlight methods for the structural characterization of cobalamin-dependent radical SAM (RS) enzymes and describe recent advances in in the overproduction and purification of these enzymes.