Fungal-derived brevianamide assembly by a stereoselective semipinacolase

Abstract

Fungal bicyclo[2.2.2]diazaoctane indole alkaloids represent an important family of natural products with a wide-spectrum of biological activities. Although biomimetic total syntheses have been completed for representative compounds, the details of their biogenesis, especially the mechanisms for assembly of diastereomerically distinct and enantiomerically antipodal metabolites, have remained largely uncharacterized. Brevianamide A represents the most basic form of the sub-family bearing a dioxopiperazine core and a rare 3-spiro-ψ-indoxyl skeleton. Here, we identified the Brevianamide A biosynthetic gene cluster from Penicillium brevicompactum NRRL 864 and fully elucidated the metabolic pathway by gene disruption, heterologous expression, precursor incorporation experiments, and in vitro biochemical analysis. In particular, we determined BvnE as a cofactor-independent isomerase/pinacolase that is essential for selective production of Brevianamide A. Structural elucidation, molecular modeling, and mutational analysis of BvnE, and quantum chemical calculations provided critical mechanistic insights into the diastereoselective formation of the 3-spiro-ψ-indoxyl moiety in Brevianamide A. This occurs through a BvnE-controlled semi-pinacol rearrangement and a subsequent spontaneous intramolecular [4+2] hetero-Diels-Alder cycloaddition. Resolution of this 50-year old mechanistic mystery together with our recent characterization of the Diels-Alderase-mediated biogenesis of monooxopiperazines highlight the diversified biosynthetic strategies deployed by fungi for creating structurally diverse spiro-cyclized indole alkaloids.Fungal indole alkaloids bearing the unusual bicyclo[2.2.2]diazaoctane core have drawn considerable attention from natural product, synthetic and biological chemists for decades. A wealth of studies on the discovery of analogs (including semi-synthetic, synthetic and natural), biological activities and biosynthetic mechanisms have been

Refactoring the Concise Biosynthetic Pathway of Cyanogramide Unveils Spirooxindole Formation Catalyzed by a P450 Enzyme

“…Two types of enzymes have been reported to catalyze spirooxindole formation in natural products biosynthesis (Figure S21). The first type is flavin‐dependent monooxygenases (FMOs), such as BvnB, FqzB, PhqK, and NotI (NotI′) in the biosynthesis of brevianamides spirotryprostatin A paraherquamides, and notoamides . FMOs were proposed to catalyze the formation of spirooxindole through an epoxide intermediate and a subsequent semipinacol‐type rearrangement.…”

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

“…[20a] Moreover, we cannot eliminate the possibility of a brevianamide A-like route similar to what we elucidated recently. [24] This would involve the sequential FMO-mediated epoxidation, P450catalyzed desaturation of the dioxopiperazine ring, and spontaneous IMDA to generate 5 or 10 without passing through 4 or 9 (Scheme 2).…”

“…[13] It is hypothesized that the C2-C3 bond of the indole is epoxidized by the FMO on the less-hindered face, as has been reported in synthetic approaches, [2d,15] and suggested for similar molecules such as the taichunamides and paraherquamides. [13,24] Protonation of the reactive epoxide intermediate leads to ring opening to form a hydroxy cation and the subsequent deprotonation facilitates the 1,2 shift to provide notoamide B (Scheme 3). While the enzymes have evolved a mechanism for stereospecific collapse of the indole epoxide, unnatural substrates seem to evade this catalyst-controlled selectivity.…”

Refactoring the Concise Biosynthetic Pathway of Cyanogramide Unveils Spirooxindole Formation Catalyzed by a P450 Enzyme

“…Two types of enzymes have been reported to catalyze spirooxindole formation in natural products biosynthesis ( Figure S21). The first type is flavin-dependent monooxygenases (FMOs), such as BvnB, FqzB, PhqK, and NotI (NotI') in the biosynthesis of brevianamides [9] spirotryprostatin A [10] paraherquamides, [11] and notoamides. [12a] FMOs were proposed to catalyze the formation of spirooxindole through an epoxide intermediate and a subsequent semipinacol-type rearrangement.…”