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The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain

Nature Structural & Molecular Biology volume 19, pages 767–772 (2012)Cite this article

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Abstract

Peptide macrocycles are found in many biologically active natural products. Their versatility, resistance to proteolysis and ability to traverse membranes has made them desirable molecules. Although technologies exist to synthesize such compounds, the full extent of diversity found among natural macrocycles has yet to be achieved synthetically. Cyanobactins are ribosomal peptide macrocycles encompassing an extraordinarily diverse range of ring sizes, amino acids and chemical modifications. We report the structure, biochemical characterization and initial engineering of the PatG macrocyclase domain of Prochloron sp. from the patellamide pathway that catalyzes the macrocyclization of linear peptides. The enzyme contains insertions in the subtilisin fold to allow it to recognize a three-residue signature, bind substrate in a preorganized and unusual conformation, shield an acyl-enzyme intermediate from water and catalyze peptide bond formation. The ability to macrocyclize a broad range of nonactivated substrates has wide biotechnology applications.

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Figure 1: Macrocyclization of patellamides.
Figure 2: Structure of PatGmac.
Figure 3: Biochemical characterization of PatGmac and PatGmac mutants.
Figure 4: Proposed mechanism for macrocyclization.

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Acknowledgements

We would like to thank J. Reeks for help with the manuscript. W.E.H. is supported as the recipient of a SULSA postdoctoral fellowship. J.K. is supported as the recipient of a DFG postdoctoral fellowship. M.C.M.S. acknowledges funding from the BBSRC project grant BB/F003439/1. Mass spectrometry is supported by the Wellcome Trust. The project is funded by the Leverhulm Trust Grant RPG-2012-504 (J.H.N. and M.J.).

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Author notes

  1. Jesko Koehnke, Andrew Bent and Wael E Houssen: These authors contributed equally to this work.

Authors and Affiliations

  1. Biomedical Sciences Research Complex, University of St Andrews, St Andrews, UK

    Jesko Koehnke, Andrew Bent, David Zollman, Falk Morawitz, Sally Shirran, Catherine H Botting & James H Naismith

  2. Department of Chemistry, Marine Biodiscovery Centre, University of Aberdeen, Aberdeen, UK

    Wael E Houssen, Ada F Nneoyiegbe, Laurent Trembleau & Marcel Jaspars

  3. Institute of Medical Sciences, School of Medicine and Dentistry, University of Aberdeen, Aberdeen, UK

    Wael E Houssen

  4. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA

    Jeremie Vendome

  5. Howard Hughes Medical Institute, Columbia University, New York, New York, USA

    Jeremie Vendome

  6. Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, Aberdeen, UK

    Margaret C M Smith

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Contributions

J.K., A.B., W.E.H. and J.H.N. carried out experiments, interpreted data and wrote the paper. D.Z., F.M., S.S., J.V., A.F.N., C.H.B. and L.T. carried out experiments and interpreted data. M.C.M.S. and M.J. interpreted data and wrote the paper.

Corresponding authors

Correspondence to Marcel Jaspars or James H Naismith.

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Competing interests

The Universities of St Andrews and Aberdeen have filed a patent application with the UK Patent Office on the use of the biosynthetic enzymes from the patellamide pathway. J.K., A.B., J.H.N., M.J., W.E.H. and M.C.M.S. are among those listed as inventors.

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Koehnke, J., Bent, A., Houssen, W. et al. The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain. Nat Struct Mol Biol 19, 767–772 (2012). https://doi.org/10.1038/nsmb.2340

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