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Plasticity, dynamics, and inhibition of emerging tetracycline resistance enzymes

Nature Chemical Biology volume 13, pages 730–736 (2017)Cite this article

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Abstract

Although tetracyclines are an important class of antibiotics for use in agriculture and the clinic, their efficacy is threatened by increasing resistance. Resistance to tetracyclines can occur through efflux, ribosomal protection, or enzymatic inactivation. Surprisingly, tetracycline enzymatic inactivation has remained largely unexplored, despite providing the distinct advantage of antibiotic clearance. The tetracycline destructases are a recently discovered family of tetracycline-inactivating flavoenzymes from pathogens and soil metagenomes that have a high potential for broad dissemination. Here, we show that tetracycline destructases accommodate tetracycline-class antibiotics in diverse and novel orientations for catalysis, and antibiotic binding drives unprecedented structural dynamics facilitating tetracycline inactivation. We identify a key inhibitor binding mode that locks the flavin adenine dinucleotide cofactor in an inactive state, functionally rescuing tetracycline activity. Our results reveal the potential of a new tetracycline and tetracycline destructase inhibitor combination therapy strategy to overcome resistance by enzymatic inactivation and restore the use of an important class of antibiotics.

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Figure 1: Dose–response curve showing the effect of tetracycline on growth of Legionella strains.
Figure 2: Crystal structures of Tet(50), Tet(51), Tet(55), and Tet(56) reveal a conserved architecture, structural changes that enable substrate-loading channel accessibility, and two conformations of the FAD cofactor.
Figure 3: Tet(50)–chlortetracycline structure reveals an unexpected mode of binding that drives substrate-loading channel closure and FAD conversion.
Figure 4: Chlortetracycline is degraded by tetracycline destructases despite the unusual binding mode.
Figure 5: Anhydrotetracycline binds to the active site of Tet(50), trapping FAD in the unproductive OUT conformation.
Figure 6: Anhydrotetracycline prevents enzymatic tetracycline degradation, functionally rescuing tetracycline antibiotic activity.

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Acknowledgements

We thank J. Nix and ALS beamline 4.2.2 (contract DE-AC02-05CH11231) for assistance with X-ray data collection and A. Durairaj of the Proteomics & Mass Spectrometry Facility at the Danforth Plant Science Center for assistance with HR–MS/MS experiments. This work was supported by an award to N.H.T., G.D., and T.A.W. from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (R01 AI123394). A.J.G. is supported by the National Institute of General Medical Sciences Cell and Molecular Biology Training Grant (T32 GM007067). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

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Author notes

  1. Jooyoung Park and Andrew J Gasparrini: These authors contributed equally to this work.

  2. Timothy A Wencewicz, Gautam Dantas and Niraj H Tolia: These authors jointly directed this work.

Authors and Affiliations

  1. Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA

    Jooyoung Park, Jennifer L Elliott, Joseph P Vogel, Gautam Dantas & Niraj H Tolia

  2. Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri, USA

    Andrew J Gasparrini & Gautam Dantas

  3. Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri, USA

    Margaret R Reck, Chanez T Symister & Timothy A Wencewicz

  4. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA

    Gautam Dantas

  5. Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, Missouri, USA

    Gautam Dantas

  6. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA

    Niraj H Tolia

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Contributions

J.P. designed and performed crystallographic experiments and X-ray structure determination, analyzed data, and wrote the paper; A.J.G. designed and performed in vitro and microbiological experiments, analyzed data, and wrote the paper; M.R.R. and C.T.S. performed in vitro experiments; J.L.E. performed crystallographic experiments; J.P.V. performed Legionella experiments; T.A.W., G.D., and N.H.T. designed experiments, analyzed data, and wrote the manuscript.

Corresponding authors

Correspondence to Timothy A Wencewicz, Gautam Dantas or Niraj H Tolia.

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Park, J., Gasparrini, A., Reck, M. et al. Plasticity, dynamics, and inhibition of emerging tetracycline resistance enzymes. Nat Chem Biol 13, 730–736 (2017). https://doi.org/10.1038/nchembio.2376

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