Genome Mining of Isoindolinone-Containing Peptide Natural Products

Peptide natural products (PNPs) are important sources of bioactive compounds. Recent studies have shown that oligopeptides or pseudopeptides can be synthesized by amide-bond-forming enzymes such as ATP-grasp enzymes and amide-bond synthetases (ABSs). By focusing on ATP-grasp enzymes as part of a conserved biosynthetic enzyme ensemble, genome mining of PNPs was performed on three biosynthetic gene clusters (BGCs) from diverse fungi, including Coccidioides immitis RS, the causative agent of valley fever. We demonstrate that the conserved enzymes synthesize a common dipeptide fragment, l-leucine-l-O-isoindolinone-homoserine (l-Leu-l-Isd), which is modified and diversified into three PNPs (1-3) by associated enzymes in the three pathways. Pathway reconstitution and enzymatic assays led to the characterization of six ATP-grasp enzymes and ABSs that catalyze di-, tri-, and tetrapeptide formation. From the C. immitis BGC, a flavoenzyme catalyzing the direct oxidation of l-tryptophan to l-oxindolylalanine was discovered. Our work validates ATP-grasp enzymes and ABSs as leads to mine new PNPs and further showcases the biocatalytic potential of these enzymes in catalyzing amide-bond formation.