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

Many biologically active natural products are synthesized by nonribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs) and their hybrids. These megasynthetases are comprised of modules which are subdivided into distinct catalytic domains that allow for substrate initiation, chain extension, processing and termination. At the end of a module, a terminal domain, usually a thioesterase (TE), is responsible for catalyzing the release of the NRPS or PKS as a linear or cyclized product. In this Review, we address the general cyclization mechanism of the TE domain, including oligomerization and the fungal CÀ C bond forming Claisen-like cyclases (CLCs).Additionally, we include examples of cyclization catalysts acting within or at the end of a module. Furthermore, condensationlike (C T ) domains, terminal reductase (R) domains, reductase-like domains that catalyze Dieckmann condensation (R D ), thioesterase-like Dieckmann cyclases, trans-acting TEs from the penicillin binding protein (PBP) enzyme family, product template (PT) domains and others will also be reviewed. The studies summarized here highlight the remarkable diversity of NRPS and PKS cyclization catalysts for the production of biologically relevant, complex cyclic natural products and related compounds.

<p>Fungal bicyclo[2.2.2]diazaoctane indole alkaloids demonstrate intriguing structures and a wide spectrum of biological activities. Although biomimetic total syntheses have been completed for representative compounds of this structural family, the details of their biogenesis have remained largely uncharacterized. Among them, Brevianamide A represents the most basic form within this class bearing a dioxopiperazine core structure and a rare 3-<i>spiro</i>-<i>psi</i>-indoxyl skeleton. Here, we identified the Brevianamide A biosynthetic gene cluster from <i>Penicillium brevicompacticum</i> NRRL 864 and fully elucidated the metabolic pathway by targeted gene disruption, heterologous expression, precursor incorporation studies, and <i>in vitro</i> biochemical analysis. In particular, we determined that BvnE is a cofactor-independent isomerase that is essential for selective production of Brevianamide A. Based on a high resolution crystal structure of BvnE, molecular modeling, mutational analysis, and computational studies provided new mechanistic insights into the diastereoselective formation of the 3-<i>spiro</i>-<i>psi</i>-indoxyl moiety in Brevianamide A. This occurs through a biocatalyst controlled semi-Pinacol rearrangement and a subsequent spontaneous intramolecular [4+2] <i>hetero</i>-Diels-Alder cycloaddition.</p>

<p>Fungal bicyclo[2.2.2]diazaoctane indole alkaloids demonstrate intriguing structures and a wide spectrum of biological activities. Although biomimetic total syntheses have been completed for representative compounds of this structural family, the details of their biogenesis have remained largely uncharacterized. Among them, Brevianamide A represents the most basic form within this class bearing a dioxopiperazine core structure and a rare 3-<i>spiro</i>-<i>psi</i>-indoxyl skeleton. Here, we identified the Brevianamide A biosynthetic gene cluster from <i>Penicillium brevicompacticum</i> NRRL 864 and fully elucidated the metabolic pathway by targeted gene disruption, heterologous expression, precursor incorporation studies, and <i>in vitro</i> biochemical analysis. In particular, we determined that BvnE is a cofactor-independent isomerase that is essential for selective production of Brevianamide A. Based on a high resolution crystal structure of BvnE, molecular modeling, mutational analysis, and computational studies provided new mechanistic insights into the diastereoselective formation of the 3-<i>spiro</i>-<i>psi</i>-indoxyl moiety in Brevianamide A. This occurs through a biocatalyst controlled semi-Pinacol rearrangement and a subsequent spontaneous intramolecular [4+2] <i>hetero</i>-Diels-Alder cycloaddition.</p>