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.
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This work was supported by the National Institutes of Health R01 CA070375 to (R.M.W. and D.H.S.), R35 GM118101 and the Hans W. Vahlteich Professorship (to D.H.S.), and R01 DK042303 and the Margaret J. Hunter Professorship (to J.L.S.). J.N.S. and K.N.H. acknowledge support from the National Institute of General Medical Sciences of the National Institutes of Health under awards F32GM122218 (to J.N.S.) and R01GM124480 (to K.N.H.). Computational resources were provided by the UCLA Institute for Digital Research and Education (IDRE) and the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the NSF (OCI-1053575). Anton 2 computer time was provided by the Pittsburgh Supercomputing Center (PSC) through grant no. R01GM116961 from the National Institutes of Health. The Anton 2 machine at PSC was generously made available by D.E. Shaw Research. GM/CA@APS is supported by the National Institutes of Health, National Institute of General Medical Sciences (AGM-12006) and National Cancer Institute (ACB-12002). We thank S. Ragsdale for assistance with anaerobic enzyme assays and P. Nagorny for assistance with polarimetry measurements.
The authors declare no competing interests.
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Dan, Q., Newmister, S.A., Klas, K.R. et al. Fungal indole alkaloid biogenesis through evolution of a bifunctional reductase/Diels–Alderase. Nat. Chem. 11, 972–980 (2019). https://doi.org/10.1038/s41557-019-0326-6
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