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

Abstract

Cyanobactins comprise a widespread group of peptide metabolites produced by cyanobacteria that are often diversified by post‐translational prenylation. Several enzymes have been identified in cyanobactin biosynthetic pathways that carry out chemically diverse prenylation reactions, representing a resource for the discovery of post‐translational alkylating agents. Here, genome mining was used to identify orphan cyanobactin prenyltransferases, leading to the isolation of tolypamide from the freshwater cyanobacterium Tolypothrix sp. The structure of tolypamide was confirmed by spectroscopic methods, degradation, and enzymatic total synthesis. Tolypamide is forward‐prenylated on a threonine residue, representing an unprecedented post‐translational modification. Biochemical characterization of the cognate enzyme TolF revealed a prenyltransferase with strict selectivity for forward O‐prenylation of serine or threonine but with relaxed substrate selectivity for flanking peptide sequences. Since cyanobactin pathways often exhibit exceptionally broad substrate tolerance, these enzymes represent robust tools for synthetic biology.

AgeMTPT, a Catalyst for Peptide N-Terminal Modification

“…AgeMTPT was expressed in Escherichia coli and purified as previously described, and all substrates were made using solid-phase peptide synthesis (SPPS) followed by purification to homogeneity. The prenyltransferase reaction conditions were as previously optimized by Sardar et al All enzymatic reactions were analyzed by UPLC-ESI-TOF-MS, following protocols that we developed for peptide prenyltransferase enzymes. ,,,− …”

Biological Dark Matter Exploration using Data Mining for the Discovery of Antimicrobial Natural Products

“…PCC 7601 as a producer of tolypamide, a cyanobactin with antimicrobial activity structurally differentiated by bearing a prenyl moiety bound to a threonine residue ( Fig. 2 ) 35 . Another example for data mining in a new taxon is the genus Micromonospora , whose members can produce analogues of ramoplanin and enduracin.…”

Chemoenzymatic Late‐Stage Modifications Enable Downstream Click‐Mediated Fluorescent Tagging of Peptides

“…Cyanobactins are a family of cyanobacterial modified linear and macrocyclic peptides that are produced through post‐translational modification of ribosomally encoded precursor peptides [1–3] . These post‐translational modifications include heterocyclization to generate thiazoline, oxazoline and methyloxazoline, [4–7] oxidation of azolines to the corresponding azoles, [8] N‐methylation of histidine, [9, 10] N‐to‐C‐macrocyclization [11–14] and forward and reverse prenylation and geranylation [15–28] . Cyanobactin prenyltransferases belong to the aromatic prenyltransferases ABBA superfamily, which are named after the αββα succession of secondary structure elements and are characterized by having a β‐barrel core that is surrounded by a ring of solvent‐exposed α‐helices [16, 29–32] .…”