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Discovery of a new ATP-binding motif involved in peptidic azoline biosynthesis

Nature Chemical Biology volume 10, pages 823–829 (2014)Cite this article

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Abstract

Despite intensive research, the cyclodehydratase responsible for azoline biogenesis in thiazole/oxazole-modified microcin (TOMM) natural products remains enigmatic. The collaboration of two proteins, C and D, is required for cyclodehydration. The C protein is homologous to E1 ubiquitin-activating enzymes, whereas the D protein is within the YcaO superfamily. Recent studies have demonstrated that TOMM YcaOs phosphorylate amide carbonyl oxygens to facilitate azoline formation. Here we report the X-ray crystal structure of an uncharacterized YcaO from Escherichia coli (Ec-YcaO). Ec-YcaO harbors an unprecedented fold and ATP-binding motif. This motif is conserved among TOMM YcaOs and is required for cyclodehydration. Furthermore, we demonstrate that the C protein regulates substrate binding and catalysis and that the proline-rich C terminus of the D protein is involved in C protein recognition and catalysis. This study identifies the YcaO active site and paves the way for the characterization of the numerous YcaO domains not associated with TOMM biosynthesis.

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Figure 1: YcaO gene clusters and characterized roles of YcaO proteins.
Figure 2: Structure of Ec-YcaO and ATP-binding residues of Ec-YcaO.
Figure 3: Conservation of the Ec-YcaO ATP-binding residues in the superfamily.
Figure 4: BalhC modulates ATP binding and hydrolysis by BalhD and is responsible for leader peptide binding.

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Acknowledgements

We are grateful to C. Deane and K. Taylor for the generation of select BalhD mutants and J. Melby for assistance with collecting MS/MS data. This work was supported by the US National Institutes of Health (NIH) (1R01 GM097142 to D.A.M., 1R01 GM102602 to S.K.N. and 2T32 GM070421 to K.L.D., B.J.B. and J.R.C.). Additional support was from the Harold R. Snyder Fellowship (University of Illinois at Urbana-Champaign (UIUC) Department of Chemistry to K.L.D.), the Robert C. and Carolyn J. Springborn Endowment (UIUC Department of Chemistry to B.J.B.), the National Science Foundation Graduate Research Fellowship (DGE-1144245 to B.J.B.) and the University of Illinois Distinguished Fellowship (UIUC Graduate College to J.R.C.) The Bruker UltrafleXtreme MALDI TOF/TOF mass spectrometer was purchased in part with a grant from the NIH–National Center for Research Resources (S10 RR027109 A).

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Author notes

  1. Kyle L Dunbar and Jonathan R Chekan: These authors contributed equally to this work.

Authors and Affiliations

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

    Kyle L Dunbar, Brandon J Burkhart & Douglas A Mitchell

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

    Kyle L Dunbar, Courtney L Cox, Brandon J Burkhart, Satish K Nair & Douglas A Mitchell

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

    Jonathan R Chekan & Satish K Nair

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

    Courtney L Cox & Douglas A Mitchell

  5. Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Satish K Nair

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Contributions

Experiments were designed by D.A.M., S.K.N., K.L.D., J.R.C., C.L.C. and B.J.B. and were performed by K.L.D., J.R.C., C.L.C. and B.J.B. The manuscript was written by D.A.M., K.L.D. and J.R.C. with critical editorial input from S.K.N., C.L.C. and B.J.B. The overall study was conceived and managed by D.A.M. with S.K.N. overseeing all aspects of protein structure determination.

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Correspondence to Satish K Nair or Douglas A Mitchell.

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Dunbar, K., Chekan, J., Cox, C. et al. Discovery of a new ATP-binding motif involved in peptidic azoline biosynthesis. Nat Chem Biol 10, 823–829 (2014). https://doi.org/10.1038/nchembio.1608

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