Prenyltransfer is an early-stage carbon–hydrogen bond (C–H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Towards the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). In the biosynthesis of cytotoxic notoamide metabolites, PT NotF is responsible for catalyzing the first tailoring event of C2 reverse prenyltransfer of brevianamide F (cyclo(L-Trp-L-Pro)). Obtaining a high-resolution crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent exposed substrate binding site, intimating NotF may possess significant substrate promiscuity. To assess the full potential of NotF’s broad substrate selectivity, we synthesized a panel of 30 tryptophanyl DKPs with a suite of sterically and electronically differentiated amino acids, which were selectively prenylated by NotF in often synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library enabled the development of a descriptive statistical model that provided insight into the origins of NotF’s substrate promiscuity. Through this unique approach for understanding enzyme scope, we identified key substrate descriptors such as electrophilicity, size, and flexibility, that govern enzymatic turnover by NotF. Additionally, we demonstrated the ability to couple NotF-catalyzed prenyltransfer with oxidative cyclization using recently characterized flavin monooxygenase, BvnB, from the brevianamide biosynthetic pathway. This one-pot, in vitro biocatalytic cascade proceeds with exceptional substrate recognition, and enabled the first chemoenzymatic synthesis of the marine fungal natural product, (–)-eurotiumin A, in three steps and 60% overall yield.
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