Rational Engineering of the Radical SAM Enzyme HtkB Expands the Chemical Space of Cyclophane RiPPs

Cyclophane-containing peptides represent a structurally diverse class of macrocycles with important applications in drug development due to their stability and target specificity. Radical S-adenosylmethionine (rSAM) enzymes are key catalysts for cyclophane formation and execute chemically challenging C(sp2)–C(sp3) crosslinks. However, rSAM cyclophane synthases have generally been restricted to acting on their native or engineered substrates. Here, we report structure-guided engineering of an rSAM cyclophane synthase through mutation of a conserved D214 residue in the rSAM enzyme HtkB. This single amino acid substitution substantially broadens substrate tolerance, enabling macrocyclization of diverse side chains, including polar, aliphatic, and acidic residues. Characterization of the His-to-Leu crosslinked product shows that C–C bond formation is analogous to the wild-type His-to-Lys crosslink and that the regiospecificity and stereospecificity of the enzyme are conserved. These findings reveal the unexpected plasticity of an rSAM cyclophane synthase and open opportunities to engineer rSAM enzymes for broader biocatalytic and therapeutic applications.