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

Abstract

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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Fig. 1: Fungal bicyclo[2.2.2]diazaoctane indole alkaloids and biosynthesis.
Fig. 2: Biomimetic synthesis of premalbrancheamide.
Fig. 3: In vitro enzymatic reconstitution of malbrancheamide biosynthesis.
Fig. 4: Structures of MalC and PhqE.
Fig. 5: Catalytic mechanism of the MalC/PhqE-catalysed Diels–Alder reaction.

Data availability

Coordinates and associated structure factors have been deposited with the PDB under accession codes 6NKH (MalC), 6NKI (PhqB RNADPH), 6NKK (PhqE1NADP+) and 6NKM (PhqE D166N11NADP+).

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Acknowledgements

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.

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Q.D., S.A.N., J.L.S., R.M.W. and D.H.S. contributed to the experimental design. Q.D., S.A.N., A.E.F. and W.C.B. performed molecular cloning, protein expression and purification. Q.D., S.A.N. and A.E.F. performed all enzymatic assays and LC/MS analysis. S.A.N. and Q.D. carried out all crystallographic experiments, structural analysis and structure-based site-directed mutagenesis. K.R.K., J.D.S., A.D.S., T.J.M., L.Z., S.A.N. and V.V.S. synthesized and validated all the compounds described in this study. Y.Y. and F.Y. carried out the genetic knockout experiment, and F.Y. and Q.D. performed genetic annotation. J.N.S. and S.A.N. performed molecular dynamics simulations. R.S.P. performed DFT calculations. Q.D., S.A.N., K.N.H., J.L.S., R.M.W. and D.H.S. evaluated the data and prepared the manuscript.

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Correspondence to David H. Sherman or Robert M. Williams.

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Methods, Figs. 1–26, Tables 1–4 (including X-ray data collection and refinement statistics) and all NMR spectra of newly synthesized compounds and references.

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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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