A Single Amino Acid Switch Alters the Isoprene Donor Specificity in Ribosomally Synthesized and Post-Translationally Modified Peptide Prenyltransferases

Abstract

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). Though most characterized RiPP prenyltransferases carry out the regiospecific transfer of C 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 donor, with no C 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 utilizing enzymes. Remarkably, only a single amino acid mutation is necessary to completely switch the donor specificity from a C to a C prenyltransferase, and vice versa. Lastly, we demonstrate that these enzymes may be used for the chemospecific attachment of C or C 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.

New developments in RiPP discovery, enzymology and engineering

“…416 Though the vast majority of RiPP PTases catalyze the regioselective transfer of C5 dimethylallyl donors to their cognate peptide substrates, reconstitution of the activity of PirF from piricyclamide in Microcystis aeruginosa conrmed its Tyr Ogeranylation activity. 425,426 In vitro reconstitution experiments elucidated that PirF specically geranylates Tyr residues when presented with Ser, Thr, Trp, or Tyr derivatives as substrates. 425 Experiments with further small molecule substrates revealed a broad substrate scope for PirF, which is able to geranylate Land D-Tyr, in addition to a selection of small phenolic compounds, though larger Tyr-containing peptide substrates are favored to small amino acids.…”

Microbial soluble aromatic prenyltransferases for engineered biosynthesis

“…In the cocrystal structure of PagF/DMSPP, the Phe222 residue is located at the top of the barrel, exerting an essential hydrophobic effect to stabilize the allylic carbocation against solvent quenching ( Fig. 12 A) [ 49 ]. Based on these findings, Phe222 mutants were investigated.…”

Genome‐Mining‐Based Discovery of the Cyclic Peptide Tolypamide and TolF, a Ser/Thr Forward O‐Prenyltransferase

“…In 2018, Estrada et al. showed that a single Phe to Gly mutation at the hydrophobic active site of PagF, shifts the donor preference from DMAPP to GPP [11b] . This is a direct consequence of active site expansion in the presence of a smaller glycine residue.…”