Molecular Basis for Spirocycle Formation in the Paraherquamide Biosynthetic Pathway

Abstract

<div> <div> <div> <p>The paraherquamides are potent anthelmintic natural products with complex heptacyclic scaffolds. One key feature of these molecules is the spiro-oxindole moiety that lends a strained three-dimensional architecture to these structures. The flavin monooxygenase PhqK was found to catalyze spirocycle formation through two parallel pathways in the biosynthesis of paraherquamides A and G. Two new paraherquamides (K and L) were isolated from a ΔphqK strain of Penicillium simplicissimum, and subsequent enzymatic reactions with these compounds generated two additional metabolites paraherquamides M and N. Crystal structures of PhqK in complex with various substrates provided a foundation for mechanistic analyses and computational studies. While it is evident that PhqK can react with various substrates, reaction kinetics and molecular dynamics simulations indicated that the dioxepin-containing paraherquamide L was the favored substrate. Through this effort, we have elucidated a key step in the biosynthesis of the paraherquamides, and provided a rationale for the selective spirocyclization of these powerful anthelmintic agents. </p></div></div><div><div> </div> </div> </div>

Enzyme evolution in fungal indole alkaloid biosynthesis

“…To probe the timing of spirocyclization and dioxepin ring formation, triply deuterium‐labeled 7‐hydroxy‐ 25 was introduced to a culture of Penicillium fellutanum; the results indicated that indole C7 oxidation is not the immediate step following the IMDA reaction, but still may occur prior to spirooxindole formation [60]. Recent gene disruption studies in Penicillium simplicissimum established that the pyran and dioxepin rings are both formed prior to spirocyclization, elucidating two key intermediates en route to paraherquamides A ( 1 ) and G ( 83 ) [61].…”

“…The PhqK flavin monooxygenase (FMO) accepts both paraherquamides K ( 26 ) and L ( 27 ) and performs a facially selective epoxidation, with a controlled collapse of the epoxide to form the spirooxindole (Fig. 2) [61]. Crystal structures revealed that substrate orientation in the PhqK active site determined the facial selectivity of epoxidation.…”

“…The semipinacol rearrangement is proposed to proceed through general base catalysis to generate the indoxyl [52]. In comparison, paraherquamide biosynthesis utilizes general acid catalysis to generate an oxindole moiety [61], demonstrating that these systems have evolved divergent mechanisms for spirocycle formation.…”

“…The halogenases MalA ( M. aurantiaca ) and MalA′ ( M. graminicola ) are responsible for the iterative chlorination to generate the natural product malbrancheamide ( 67 ) [4,78,79]. These halogenases catalyze chlorination and bromination reactions to generate monochlorinated malbrancheamide B ( 68 ) and isomalbrancheamide B ( 69 ), monobrominated malbrancheamide C ( 70 ) and isomalbrancheamide C ( 71 ), mixed halogen malbrancheamide D ( 72 ) and isomalbrancheamide D ( 73 ), and the dibrominated malbrancheamide E [61] (Fig. 15B).…”