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Structural basis for macrolactonization by the pikromycin thioesterase

Abstract

Polyketides are a class of biologically active microbial and plant-derived metabolites that possess a high degree of structural and functional diversity and include many human therapeutics, among them anti-infective and anti-cancer drugs, growth promoters and anti-parasitic agents1. The macrolide antibiotics, characterized by a glycoside-linked macrolactone, constitute an important class of polyketides, including erythromycin and the natural ketolide anti-infective agent pikromycin. Here we describe new mechanistic details of macrolactone ring formation catalyzed by the pikromycin polyketide synthase thioesterase domain from Streptomyces venezuelae. A pentaketide phosphonate mimic of the final pikromycin linear chain-elongation intermediate was synthesized and shown to be an active site affinity label. The crystal structures of the affinity-labeled enzyme and of a 12-membered-ring macrolactone product complex suggest a mechanism for cyclization in which a hydrophilic barrier in the enzyme and structural restraints of the substrate induce a curled conformation to direct macrolactone ring formation.

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Figure 1: Affinity label adduct and macrolactone product within the Pik TE active site.
Figure 2: Electrostatic surface representation of Pik TE substrate channel with affinity label and with macrolactone product.
Figure 3: Model of acyl-serine hepta- and hexaketides in the active site shown from the top views (left; orientation is similar to that in Fig. 1b) and side views (right).

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Acknowledgements

This research was generously supported by grants from the University of Minnesota Graduate School (to R.A.F.), by grants DK042303 (to J.L.S.) and GM076477 (to D.H.S.) from the US National Institutes of Health (NIH). J.D.K. was supported by the Hans and Ella McCollum Vahlteich Research Fund at the University of Michigan College of Pharmacy and an NIH postdoctoral fellowship (GM075641). The authors thank L. Venkatraman for the synthesis of aldehyde 15 and J. Konwerski for expert assistance with protein purification and crystallization. The GM/CA Collaborative Access Team facility at the APS is supported by the NIH Institute of General Medical Sciences (GM) and National Cancer Institute (CA). Use of the APS was supported by the US Department of Energy.

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Authors and Affiliations

Authors

Contributions

D.L.A., J.D.K. and J.L.S. were responsible for the Pik TE production, crystallography and structural analysis; J.W.G. and R.A.F. were responsible for the design and synthesis of the diphenylphosphonate pentaketide; and J.D.K. and D.H.S. were responsible for conducting the Pik TE inactivation experiments.

Note: Supplementary information is available on the Nature Chemical Biology website.

Corresponding authors

Correspondence to Robert A Fecik, David H Sherman or Janet L Smith.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Time course of Pik TE inactivation by reduced pentaketide 11. (PDF 14 kb)

Supplementary Fig. 2

Stereodiagram of simulated-annealing FoFc omit maps. (PDF 3945 kb)

Supplementary Table 1

Data collection and refinement statistics. (PDF 54 kb)

Supplementary Scheme 1

Synthesis of diphenylphosphonate pentaketide 11. (PDF 55 kb)

Supplementary Methods (PDF 853 kb)

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Akey, D., Kittendorf, J., Giraldes, J. et al. Structural basis for macrolactonization by the pikromycin thioesterase. Nat Chem Biol 2, 537–542 (2006). https://doi.org/10.1038/nchembio824

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