Functional and Structural Studies on the Esperamicin Thioesterase and Progress toward Understanding Enediyne Core Biosynthesis

Authors

• Erome D. Hankore, University of KentuckyFollow
• Mitchell D. Miller, Rice University
• Abigael J. Kosgei, Rice University
• Weijun Xu, Rice University
• Kemin Tan, Argonne National Laboratory
• Michael Endres, Argonne National Laboratory
• Minakshi Bhardwaj, University of KentuckyFollow
• Grazyna Joachimiak, Argonne National Laboratory
• Andrzej Joachimiak, Argonne National Laboratory
• George N. Phillips Jr., Rice University
• Jon S. Thorson, University of KentuckyFollow
• Steven G. Van Lanen, University of KentuckyFollow

Abstract

Enediynes are among the most potent antitumor and antibacterial natural products. Studies on their biosynthetic pathways have identified a shared, linear polyene precursor generated from an iterative type I polyketide synthase (PKSE) as the source of the enediyne warhead. A key step is the release of this polyene from the PKSE by a discrete thioesterase (TE). Here, we used X-ray crystallography, site-directed mutagenesis, and heterologous coexpression of PKSEs and TEs to elucidate how enediyne TEs mediate the production of the polyene. We solved the structure of wild-type EspE7 from esperamicin producer Actinomodura verrucosospora. The substrate binding pocket was also defined upon serendipitous cocrystallization of an EspE7 mutant with a fatty acyl-CoA ligand. Structural data and in vitro activity assays with EspE7 mutants provide strong evidence that Glu68 in EspE7 and the analogous Glu residue in other enediyne TEs functions as a key catalytic residue, thus supporting a hydrolysis mechanism for enediyne TEs that aligns with that of Pseudomonas sp. 4-HB-CoA TE. Furthermore, combinations of 9- and 10-membered enediyne PKSEs and TEs produced 1,3,5,7,9,11,13-pentadecaheptaene (1) as the major product. Thus, the data further support previous conclusions that 1 serves as the sole precursor for the biosynthesis of all enediyne cores.

Document Type

Article

Publication Date

2025

Notes/Citation Information

Copyright © 2025 The Authors. Published by American Chemical Society and American Society of Pharmacognosy.

Digital Object Identifier (DOI)

https://doi.org/10.1021/acs.jnatprod.5c00660

Funding Information

National Institutes of Health Grants U01GM098248 (to G.N.P.), R01GM115261 (J.S.T. and G.N.P.), and R01CA217255 (J.S.T., G.N.P., and S.G.V.L.); the Center of Biomedical Research Excellence (COBRE) in Pharmaceutical Research and Innovation Grant P20GM130456 (J.S.T. and S.G.V.L.); the National Science Foundation BioXFEL Science and Technology Center (Grant 1231306 (G.N.P.); and the Robert A. Welch Foundation Grant C-2118 (G.N.P).

Repository Citation

Hankore, Erome D.; Miller, Mitchell D.; Kosgei, Abigael J.; Xu, Weijun; Tan, Kemin; Endres, Michael; Bhardwaj, Minakshi; Joachimiak, Grazyna; Joachimiak, Andrzej; Phillips, George N. Jr.; Thorson, Jon S.; and Van Lanen, Steven G., "Functional and Structural Studies on the Esperamicin Thioesterase and Progress toward Understanding Enediyne Core Biosynthesis" (2025). Pharmaceutical Sciences Faculty Publications. 224.
https://uknowledge.uky.edu/ps_facpub/224