Data Science-Driven Analysis of Substrate-Permissive Diketopiperazine Reverse Prenyltransferase NotF: Applications in Protein Engineering and Cascade Biocatalytic Synthesis of (−)-Eurotiumin A

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. Toward 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). As the first tailoring event within the biosynthesis of cytotoxic
notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer
of brevianamide F. Solving a crystal structure of NotF (in complex
with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate
(DMSPP)) revealed a large, solvent-exposed active site, intimating
NotF may possess a significantly broad substrate scope. To assess
the substrate selectivity of NotF, we synthesized a panel of 30 sterically
and electronically differentiated tryptophanyl DKPs, the majority
of which were selectively prenylated by NotF in synthetically useful
conversions (2 to >99%). Quantitative representation of this substrate
library and development of a descriptive statistical model provided
insight into the molecular origins of NotF’s substrate promiscuity.
This approach enabled the identification of key substrate descriptors
(electrophilicity, size, and flexibility) that govern the rate of
NotF-catalyzed prenyltransfer, and the development of an “induced
fit docking (IFD)-guided” engineering strategy for improved
turnover of our largest substrates. We further demonstrated the utility
of NotF in tandem with oxidative cyclization using flavin monooxygenase,
BvnB. This one-pot, in vitro biocatalytic cascade
enabled the first chemoenzymatic synthesis of the marine fungal natural
product, (−)-eurotiumin A, in three steps and 60% overall yield.