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YcaO domains use ATP to activate amide backbones during peptide cyclodehydrations

Nature Chemical Biology volume 8, pages 569–575 (2012)Cite this article

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Abstract

Thiazole/oxazole-modified microcins (TOMMs) encompass a recently defined class of ribosomally synthesized natural products with a diverse set of biological activities. Although TOMM biosynthesis has been investigated for over a decade, the mechanism of heterocycle formation by the synthetase enzymes remains poorly understood. Using substrate analogs and isotopic labeling, we demonstrate that ATP is used to directly phosphorylate the peptide amide backbone during TOMM heterocycle formation. Moreover, we present what is to our knowledge the first experimental evidence that the D-protein component of the heterocycle-forming synthetase (YcaO/domain of unknown function 181 family member), formerly annotated as a docking protein involved in complex formation and regulation, is able to perform the ATP-dependent cyclodehydration reaction in the absence of the other TOMM biosynthetic proteins. Together, these data reveal the role of ATP in the biosynthesis of azole and azoline heterocycles in ribosomal natural products and prompt a reclassification of the enzymes involved in their installation.

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Figure 1: TOMM biosynthetic gene clusters and possible mechanisms of ATP use during azole formation.
Figure 2: Minimal requirement for cyclodehydratase activity and ATP-azole stoichiometry for substrate processing.
Figure 3: ATP is used by BalhD to directly activate the amide backbone of the substrate.
Figure 4: TOMM azoline installation is reminiscent of intein-mediated protein splicing.

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Acknowledgements

We are grateful to W. van der Donk for technical advice and for suggestions in the preparation of the manuscript. We thank members of the Mitchell lab and M. Marletta for critical review of this manuscript. This work was supported in part by the institutional funds provided by the University of Illinois and the US National Institutes of Health (NIH) (1R01 GM097142 to D.A.M). D.A.M. is also the recipient of the NIH Director's New Innovator Award (DP2 OD008463). K.L.D. was supported by the NIH Training Program in the Chemistry-Biology Interface (2T32 GM070421). J.O.M. was supported by the University of Illinois Department of Chemistry Chinoree T. Kimiyo Enta Fellowship.

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Authors and Affiliations

  1. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Kyle L Dunbar, Joel O Melby & Douglas A Mitchell

  2. Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Kyle L Dunbar, Joel O Melby & Douglas A Mitchell

  3. Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Douglas A Mitchell

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  1. Kyle L Dunbar
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Contributions

Experiments were designed by D.A.M., K.L.D. and J.O.M. and were performed by K.L.D. and J.O.M. The manuscript was written by D.A.M. and K.L.D. with critical editorial input from J.O.M. The study was conceived and overseen by D.A.M.

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Correspondence to Douglas A Mitchell.

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Dunbar, K., Melby, J. & Mitchell, D. YcaO domains use ATP to activate amide backbones during peptide cyclodehydrations. Nat Chem Biol 8, 569–575 (2012). https://doi.org/10.1038/nchembio.944

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