Open AccessFungal indole alkaloid biogenesis through evolution of a bifunctional reductase/Diels–Alderase
Author(s) -
Qingyun Dan,
Sean A. Newmister,
Kimberly R. Klas,
Amy E. Fraley,
Timothy McAfoos,
Amber D. Somoza,
James D. Sunderhaus,
Ying Ye,
Vikram V. Shende,
Fei Yu,
Jacob N. Sanders,
William Clay Brown,
Le Zhao,
Robert S. Paton,
K. N. Houk,
Janet L. Smith,
David H. Sherman,
Robert M. Williams
Publication year - 2019
Publication title -
nature chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.996
H-Index - 232
eISSN - 1755-4349
pISSN - 1755-4330
DOI - 10.1038/s41557-019-0326-6
Subject(s) - chemistry , stereochemistry , enantioselective synthesis , bifunctional , indole test , biosynthesis , nonribosomal peptide , combinatorial chemistry , enzyme , biochemistry , catalysis
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.