Cyclodipeptide synthases (CDPSs) constitute a family of peptide bond–forming enzymes that use aminoacyl-tRNAs for the synthesis of cyclodipeptides. Here, we describe the activity of 41 new CDPSs. We also show that CDPSs can be classified into two main phylogenetically distinct subfamilies characterized by specific functional subsequence signatures, named NYH and XYP. All 11 previously characterized CDPSs belong to the NYH subfamily, suggesting that further special features may be yet to be discovered in the other subfamily. CDPSs synthesize a large diversity of cyclodipeptides made up of 17 proteinogenic amino acids. The identification of several CDPSs having the same specificity led us to determine specificity sequence motifs that, in combination with the phylogenetic distribution of CDPSs, provide a first step toward being able to predict the cyclodipeptides synthesized by newly discovered CDPSs. The determination of the activity of ten more CDPSs with predicted functions constitutes a first experimental validation of this predictive approach.

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This work was supported by the CEA, the CNRS, the Paris-Sud University and a grant from the French National Research Agency (ANR 2010/Blan 1501 01) to M.G. and J.-L.P. I.B.J. was supported by a doctoral fellowship from the CEA. The Service d'Ingénierie Moléculaire des Protéines is member of the Laboratory of Excellence LERMIT. We warmly thank V. Dive, head of the Service d'Ingénierie Moléculaire des Protéines, for his continuous support and encouragement throughout this work. We thank O. Lespinet for advice about the building of phylogenetic trees, D. Vallenet, M. Stam and M. Sorokina for helpful discussion on bioinformatics, and A. Ponties for technical assistance in cloning experiments. We are indebted to L. Beuvier for skillful assistance in mass data analysis. We thank F. Fenaille for kindly performing the experiments using the Orbitrap mass spectrophotometer. We thank P. Kessler and O. Lequin for skillful assistance in NMR experiments.

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Author notes

    • Isabelle B Jacques
    • , Mireille Moutiez
    • , Jérôme Seguin
    • , Emmanuel Favry
    • , Muriel Gondry
    •  & Pascal Belin

    Present address: CEA, iBiTec-S, Service de Biologie Intégrative et Génétique Moléculaire, UMR 9198, Gif-sur-Yvette, France.

    • Mireille Moutiez
    •  & Jerzy Witwinowski

    These authors contributed equally to this work.


  1. Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie et de Technologies de Saclay (iBiTec-S), Service d'Ingénierie Moléculaire des Protéines, Gif-sur-Yvette, France.

    • Isabelle B Jacques
    • , Mireille Moutiez
    • , Jérôme Seguin
    • , Emmanuel Favry
    • , Robert Thai
    • , Alain Lecoq
    • , Steven Dubois
    • , Muriel Gondry
    •  & Pascal Belin
  2. Université Paris-Sud, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 8621, Institut de Génétique et Microbiologie, Orsay, France.

    • Jerzy Witwinowski
    • , Emmanuelle Darbon
    • , Cécile Martel
    •  & Jean-Luc Pernodet


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M.G. obtained funding. M.M., J.-L.P., M.G. and P.B. developed the hypothesis and designed the study. I.B.J., M.M. and P.B. performed bioinformatic analyses. I.B.J., J.W., E.D. and C.M. performed cloning experiments and prepared culture supernatants. I.B.J. performed LC/MS/MS analysis. I.B.J. and M.M. analyzed MS/MS data. I.B.J., J.S., E.F. and P.B. purified cyclodipeptides from culture supernatants. S.D. performed amino acid composition analyses. A.L. and S.D. chemically synthesized cyclodipeptides. R.T. performed high-resolution mass spectrometry. I.B.J., M.M., J.W., E.D., J.S., J.L.P., M.G. and P.B. analyzed and discussed the results. I.B.J., M.M., M.G. and P.B. prepared the draft manuscript. All of the authors participated in the production of the final version of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Muriel Gondry or Pascal Belin.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–7.

  2. 2.

    Supplementary Data Set 5

    Multiple sequence alignment of the 49 selected CDPSs plus the 11 biochemically characterized CDPSs (FASTA format). Inactive CDPSs are identified by an asterisk.

  3. 3.

    Supplementary Data Set 8

    Phylogenetic tree of CDPS sequences retrieved by database searches (September 2014).

Excel files

  1. 1.

    Supplementary Data Set 1

    Database information and sequence data relative to each putative CDPS produced in this study.

  2. 2.

    Supplementary Data Set 2

    Cyclodipeptides detected in bacterial culture supernatants.

  3. 3.

    Supplementary Data Set 3

    Sequence identity between the 11 previously identified CDPSs and the 49 CDPSs produced in this study. Sequence identities were obtained from the percent identity matrix created with Clustal Omega.

  4. 4.

    Supplementary Data Set 4

    HHPred results for the selected CDPSs.

  5. 5.

    Supplementary Data Set 6

    N1-N72 bases of available tRNAs of the organisms containing the CDPSs studied.

  6. 6.

    Supplementary Data Set 7

    Prediction of aminoacyl-binding pockets in CDPSs.

  7. 7.

    Supplementary Data Set 9

    CDPS groups according to clustering on the phylogenetic tree and identity between the P1 and P2 binding pockets.

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