| Popis: |
Mutation at a single amino acid alters the isoprene donor specificity of prenyltransferases involved in the modification of ribosomally synthesized and post-translationally modified peptides (RiPPs). While most characterized RiPP prenyltransferases carry out the regiospecific transfer of C(5) dimethylallyl donor to the side chain atoms on macrocyclic acceptor substrates, the elucidation of the cyanobactin natural product piricyclamide 70005E1 identifies an O-geranyl modification on Tyr, a reaction with little prior biochemical precedence. Reconstitution and kinetic studies of the presumptive geranyltransferase PirF shows that the enzyme utilizes a C(10) donor, with no C(5) transferase activity. The crystal structure of PirF reveals a single amino acid difference in the vicinity of the isoprene-binding pocket, relative to the C(5) utilizing enzymes. Remarkably, only a single amino acid mutation is necessary to completely switch the donor specificity from a C(5) to a C(10) prenyltransferase, and vice versa. Lastly, we demonstrate that these enzymes may be used for the chemospecific attachment of C(5) or C(10) lipid groups on lanthipeptides, an unrelated class of RiPP natural products. These studies represent a rare example where prenyl donor specificity can be discretely altered, which expands the arsenal of synthetic biology tools for tuning biological activities of peptide natural products. |
