Malbrancheamide is a dichlorinated fungal indole alkaloid isolated from both Malbranchea aurantiaca and Malbranchea graminicola that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core. The introduction of chlorine atoms on the indole ring of malbrancheamide differentiates it from other members of this family and contributes significantly to its biological activity. In this study, we characterized the two flavin-dependent halogenases involved in the late-stage halogenation of malbrancheamide in two different fungal strains. MalA and MalA’ catalyze the iterative dichlorination and monobromination of the free substrate premalbrancheamide as the final steps in the malbrancheamide biosynthetic pathway. Two unnatural bromo-chloro-malbrancheamide analogs were generated through MalA-mediated chemoenzymatic synthesis. Structural analysis and computational studies of MalA’ in complex with three substrates revealed that the enzyme represents a new class of zinc-binding flavin-dependent halogenases, and provides new insights into a potentially unique reaction mechanism.

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

Various fungi of the genera Aspergillus, Penicillium, and Malbranchea produce prenylated indole alkaloids possessing a bicyclo[2.2.2]diazaoctane ring system. After the discovery of distinct enantiomers of the natural alkaloids stephacidin A and notoamide B, from A. protuberus MF297-2 and A. amoenus NRRL 35660, another fungi, A. taichungensis, was found to produce their diastereomers, 6-epi-stephacidin A and versicolamide B, as major metabolites. Distinct enantiomers of stephacidin A and 6-epi-stephacidin A may be derived from a common precursor, notoamide S, by enzymes that form a bicyclo[2.2.2] diazaoctane core via a putative intramolecular hetero-Diels–Alder cycloaddition. This review provides our current understanding of the structural and stereochemical homologies and disparities of these alkaloids. Through the deployment of biomimetic syntheses, whole-genome sequencing, and biochemical studies, a unified biogenesis of both the dioxopiperazine and the monooxopiperazine families of prenylated indole alkaloids constituted of bicyclo[2.2.2]diazaoctane ring systems is presented.

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.

Prenylated indole alkaloids isolated from various fungi possess great structural diversity and pharmaceutical utility. Among them are the calmodulin inhibitory malbrancheamides and paraherquamides, used as anthelmintics in animal health. Herein, we report complete elucidation of the malbrancheamide biosynthetic pathway accomplished through complementary approaches. These include a biomimetic total synthesis to access the natural alkaloid and biosynthetic intermediates in racemic form, and in vitro enzymatic reconstitution that provides access to the natural antipode (+)-malbrancheamide. Reductive cleavage of a L-Pro-L-Trp dipeptide from the MalG nonribosomal peptide synthetase (NRPS) followed by reverse prenylation and a cascade of post-NRPS reactions culminates in an intramolecular [4+2] hetero-Diels-Alder (IMDA) cyclization to furnish the bicyclo[2.2.2]diazaoctane scaffold. Enzymatic assembly of optically pure (+)-premalbrancheamide involves an unexpected zwitterionic intermediate where MalC catalyzes enantioselective cycloaddition as a bifunctional NADPHdependent reductase/Diels-Alderase. Crystal structures of substrate and product complexes together with site-directed mutagenesis and molecular dynamics simulations demonstrated how MalC and PhqE, its homolog from the paraherquamide pathway, catalyze diastereo-and enantioselective cyclization in the construction of this fascinating class of secondary metabolites.

Bacterial multimodular polyketide synthases (PKSs) are large enzymatic assembly lines that synthesize many bioactive natural products of therapeutic relevance. While PKS catalysis is mostly based on fatty acid biosynthetic principles, polyketides can be further diversified by post‐PKS enzymes. Here, we characterized a remarkably versatile trans‐acyltransferase (trans‐AT) PKS from Serratia that builds structurally complex macrolides via more than ten functionally distinct PKS modules. In the oocydin PKS, we identified a new oxygenation module that α‐hydroxylates polyketide intermediates, a halogenating module catalyzing backbone γ‐chlorination, and modular O‐acetylation by a thioesterase‐like domain. These results from a single biosynthetic assembly line highlight the expansive biochemical repertoire of trans‐AT PKSs and provide diverse modular tools for engineered biosynthesis from a close relative of E. coli.

Halogenation is commonly used in medicinal chemistry to improve the potency of pharmaceutical leads. While synthetic methods for halogenation present selectivity and reactivity challenges, halogenases have evolved over time to perform selective reactions under benign conditions. The optimization of halogenation biocatalysts has utilized enzyme evolution and structure-based engineering alongside biotransformation in a variety of systems to generate stable site-selective variants. The recent improvements in halogenase-catalyzed reactions has demonstrated the utility of these biocatalysts for industrial purposes, and their ability to achieve a broad substrate scope implies a synthetic tractability with increasing relevance in medicinal chemistry.

Prenylated indole alkaloids isolated from various fungi possess great structural diversity and pharmaceutical utility. Among them are the calmodulin inhibitory malbrancheamides and paraherquamides, used as anthelmintics in animal health. Herein, we report complete elucidation of the malbrancheamide biosynthetic pathway accomplished through complementary approaches. These include a biomimetic total synthesis to access the natural alkaloid and biosynthetic intermediates in racemic form, and in vitro enzymatic reconstitution that provides access to the natural antipode (+)-malbrancheamide. Reductive cleavage of a L-Pro-L-Trp dipeptide from the MalG nonribosomal peptide synthetase (NRPS) followed by reverse prenylation and a cascade of post-NRPS reactions culminates in an intramolecular [4+2] hetero-Diels-Alder (IMDA) cyclization to furnish the bicyclo[2.2.2]diazaoctane scaffold. Enzymatic assembly of optically pure (+)-premalbrancheamide involves an unexpected zwitterionic intermediate where MalC catalyzes enantioselective cycloaddition as a bifunctional NADPH-dependent reductase/Diels-Alderase. Crystal structures of substrate and product complexes together with site-directed mutagenesis and molecular dynamics simulations demonstrated how MalC and PhqE, its homolog from the paraherquamide pathway, catalyze diastereo- and enantioselective cyclization in the construction of this important class of secondary metabolites.