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

Abstract

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.

Repurposing Modular Polyketide Synthases and Non-ribosomal Peptide Synthetases for Novel Chemical Biosynthesis

“…Finally, the growing non-ribosomal peptide chain is cleaved by four representative types of offloading domains in termination modules, producing different products including linear peptides, macrocyclic peptides, aldehydes, and tetramate moieties. The terminal X group of the product from terminal R domain includes hydroxyl group (-OH), aldehyde group (-CHO), and other aldehyde derivatives (Barajas et al, 2015;Dan et al, 2019). (B) Mechanism of polyketide chain extension for the elongation module n .…”

Artificial Enzymes for Diels‐Alder Reactions

“…Interestingly, MaDA exhibits substrates promiscuity towards both dienes and dienophiles, offering opportunities for enzyme evolution towards the utilization of non-natural substrates. X-ray structures of several natural DAases were reported (LepI, [43] MaIC, [44] Spnf, [45] AbyU, [46] PyrI4, [47] and PyrE3 [48] ,…”

“…X‐ray structures of several natural DAases were reported (LepI, [43] MaIC, [44] Spnf, [45] AbyU, [46] PyrI4, [47] and PyrE3, [48] MaDA [42] ). In most cases, 1 X‐ray structures indicate that these enzymes act as entropy traps [49] binding the substrates into a hydrophobic pocket and forcing the diene and dienophile to adopt a conformation close to the one of the TS.…”

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

“…[14] The notoamides are a fascinating class of anticancer metabolites in the indole alkaloid family where two phylogenetically related fungal strains have evolved to generate terminal products based on catalytic processes with opposing enantioselectivity (Scheme 1). The natural products (À )-stephacidin A (4), (+)-notoamide B (5), (+)-6-epi-stephacidin A (6), (À )-6-epi-stephacidin A (7), and (+)-versicolamide B (8) are produced by the terrestrial strain Aspergillus amoenus (formerly Aspergillus versicolor NRRL 35600), while (+)-stephacidin A (9), (À )-notoamide B (10), (+)-6-epi-stephacidin A (6), and (+)-versicolamide B (8) are produced by the marine strain Aspergillus protuberus (formerly Aspergillus sp. MF297-2).…”