Fungal indole alkaloid biogenesis through evolution of a bifunctional reductase/Diels-Alderase

Prenylated indole alkaloids such as the calmodulin-inhibitory malbrancheamides and anthelmintic paraherquamides possess great structural diversity and pharmaceutical utility. Here, 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 to provide access to the natural antipode (+)-malbrancheamide. Reductive cleavage of an L-Pro–L-Trp dipeptide from the MalG non-ribosomal 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 catalyses enantioselective cycloaddition as a bifunctional NADPH-dependent reductase/Diels–Alderase. The crystal structures of substrate and product complexes together with site-directed mutagenesis and molecular dynamics simulations demonstrate how MalC and PhqE (its homologue from the paraherquamide pathway) catalyse diastereo- and enantioselective cyclization in the construction of this important class of secondary metabolites

Fungal-derived brevianamide assembly by a stereoselective semipinacolase

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 of representative compounds have been reported, the details of their biogenesis, especially the mechanisms for the assembly of diastereomerically distinct and enantiomerically antipodal metabolites, have remained largely uncharacterized. Brevianamide A represents a basic form of the subfamily bearing a dioxopiperazine core and a rare 3-spiro-ψ-indoxyl skeleton. In this study, we have identified the brevianamide A biosynthetic gene cluster from Penicillium brevicompactum NRRL 864 and elucidated the metabolic pathway. BvnE was revealed to be an essential isomerase/semipinacolase that specifies the selective production of the natural product. Structural elucidation, molecular modelling and mutational analysis of BvnE as well as quantum chemical calculations have provided mechanistic insights into the diastereoselective formation of the 3-spiro-ψ-indoxyl moiety in brevianamide A. This occurs through a BvnE-controlled semipinacol rearrangement and a subsequent spontaneous intramolecular [4+2] hetero-Diels–Alder cycloaddition