Fungal Indole Alkaloid Biogenesis Through Evolution of a Bifunctional Reductase/Diels-Alderase

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.