Sequence-function space of radical SAM cyclophane synthases reveal conserved active site residues that influence substrate specificity

Radical SAM cyclophane synthases catalyze C-C, C-N, and C-O crosslinking reactions in the biosynthesis of bioactive peptide natural products. Here, we studied an uncharacterized rSAM enzyme, HtkB from Pandoraea sp., and found this enzyme to catalyze the formation of a HisC2-to-LysC beta crosslink. We used a combination of ColabFold and mutagenesis studies to show that residues D214 in HtkB and H204 in HaaB (another cyclophane synthase) are important for substrate specificity. Mutation of these residues changes the specificity and lowers substrate recognition on the wild-type motifs. This result opens opportunities to alter the specificity and promiscuity for rSAM peptide modifying enzymes. The uncharacterized rSAM enzyme HtkB from Pandoraea sp. catalyzes a HisC2-to-LysC beta crosslink. ColabFold and mutagenesis was used to reveal residues D214 in HtkB and H204 in HaaB are important for substrate specificity.