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 <i>Coccidioides immitis</i> RS, the causative agent of valley fever. We demonstrate that the conserved enzymes synthesize a common dipeptide fragment, l-leucine-l-<i>O</i>-isoindolinone-homoserine (l-Leu-l-Isd), which is modified and diversified into three PNPs (<b>1</b>-<b>3</b>) 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 <i>C. immitis</i> 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.