Mechanism and Evolution of [4+2] Cyclases in Monoterpene Indole Alkaloid Biosynthesis

Abstract

Monoterpene indole alkaloids (MIAs) comprise a large and diverse class of plant natural products, many of which exhibit potent biological activities such as anticancer, antimalarial, and antihypertensive. While the biosynthetic construction of these compounds has been a subject of intensive study for the past six decades, the identification of specific pathway genes remains an active area of investigation. MIAs belonging to the aspidosperma and iboga structural classes are produced only by select plants in the Apocynaceae (dogbane) family. The most well‐known of these is Catharanthus roseus, which produces the bisindole anticancer compounds vinblastine and vincristine through a complex pathway involving more than thirty individual enzymatic steps. Recently, we characterized three carboxylesterase‐like enzymes from C. roseus and Tabernanthe iboga that catalyze regio‐ and enantiodivergent [4+2] cycloadditions to generate the aspidosperma (tabersonine synthase, TS) and iboga (catharanthine synthase, CS; coronaridine synthase, CorS) scaffolds from a common biosynthetic intermediate. While crystal structures, computational docking, and site‐directed mutagenesis provided some key insights into the catalytic mechanism and cyclization specificity of these enzymes, several important questions remained unanswered. Here, we use a combination of computation and experiment to demonstrate that the [4+2] cyclases in MIA biosynthesis employ a stepwise rather than a concerted mechanism of cyclization. We also isolate and characterize the direct product of the CorS reaction and show that it can serve as a branch point in the biosynthesis of both iboga and pseudo‐aspidosperma alkaloids. Using a phylogenetic approach, we subsequently explore the evolution and diversification of MIA cyclase function. Reconstruction and functional analysis of ancestral enzymes allows us to trace an evolutionary trajectory from carboxylesterase to TS, from TS to CorS, and from CorS to CS. We further generate and test a panel of mutants based on both extant and ancestral cyclases to probe the molecular basis for differential cyclization activity. Our results provide insight into the mechanism and evolution of a unique class of biosynthetic enzymes and offer a means by which they could be leveraged for the facile production of new MIAs.