Molecular Basis for Spirocycle Formation in the Paraherquamide Biosynthetic Pathway

Abstract

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 threedimensional 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.

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 semi-pinacol 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 0 (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).…”

Flavin‐Dependent Monooxygenases NotI and NotI′ Mediate Spiro‐Oxindole Formation in Biosynthesis of the Notoamides

“…Very few enzymes responsible for this type of reaction within the bicyclo-ring containing family have been characterized. [12,13] NotB catalyzes 2,3-β-face epoxidation of notoamide E (1) to generate the nonspirocyclized terminal metabolites notoamides C (2) and D (3) that do not undergo IMDA cyclization. [12] Additionally, biochemical and structural analysis of PhqK provided the first insights into the mechanism of selective spirocyclization.…”