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

preprint
submitted on 24.12.2018 and posted on 26.12.2018 by Qingyun Dan, Sean A. Newmister, Kimberly R. Klas, Amy E. Fraley, Timothy J. McAfoos, Amber D. Somoza, James D. Sunderhaus, Ying Ye, Vikram V. Shende, Fengan Yu, Jacob N. Sanders, W. Clay Brown, Le Zhao, Robert S. Paton, K. N. Houk, Janet L. Smith, David H. Sherman, Robert M. Williams
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.

Funding

Total Synthesis and Biosynthesis of Bioactive Substances

National Cancer Institute

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Discovery and Characterization of Natural Product Systems

National Institute of General Medical Sciences

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Structural Biology of Complex Enzymes

National Institute of Diabetes and Digestive and Kidney Diseases

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Development of New Methods for Computational Enzyme Design

National Institute of General Medical Sciences

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Steroselectivity of Synthetically Valuable Enzyme Catalysts

National Institute of General Medical Sciences

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XSEDE: eXtreme Science and Engineering Discovery Environment

Directorate for Computer & Information Science & Engineering

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Breakthrough Molecular Dynamics Research via an Anton2 Supercomputer

National Institute of General Medical Sciences

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History

Email Address of Submitting Author

davidhs@umich.edu

Institution

University of Michigan

Country

USA

ORCID For Submitting Author

0000-0001-8334-3647

Declaration of Conflict of Interest

No competing financial interest.

Version Notes

Initial submission.

Exports