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Engineering cyclophilin into a proline-specific endopeptidase

Nature volume 391, pages 301–304 (1998)Cite this article

An Erratum to this article was published on 16 July 1998

Abstract

Designing an enzyme requires, among a number of parameters, the appropriate positioning of catalytic machinery within a substrate-binding cleft. Using the structures of cyclophilin–peptide complexes1,2,3,4, we have engineered a new catalytic activity into an Escherichia coli cyclophilin by mutating three amino acids, close to the peptide binding cleft, to form a catalytic triad similar to that found in serine proteases. In conjunction with cyclophilin's specificity for proline-bearing peptides, this creates a unique endopeptidase, cyproase 1, which cleaves peptides on the amino-side of proline residues. When acting on an Ala-Pro dipeptide, cyproase 1 has an efficiency (kcat/Km) of 0.7 × 104 M−1 s−1 and enhances the rate of reaction (kcat/kuncat) 8× 108-fold. This activity depends upon a deprotonated histidine and is inhibited by nucleophile-specific reagents, as occurs in natural serine proteases. Cyproase 1 can hydrolyse a protein substrate with a proline-specific endoprotease activity.

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Figure 1: Enlarged view of the active site of an E. coli cyclophilin (PDB entry 1LOP2) illustrating the location of the Ala-Pro binding site and the enzyme residues selected for subsequent substitutions.
Figure 2: Degradation of a protein by cyproase I.
Figure 3: pH-rate profile of cyproase I.

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Acknowledgements

We thank J. Janin, F. Lederer and G. Robillard for useful and critical reading of the manuscript.

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Authors and Affiliations

  1. Département d'Ingénierie et d'Etudes des Protines, CEA Saclay, 91191, Gif-sur-Yvette, France

    Eric Quéméneur, Mireille Moutiez, Jean-Baptiste Charbonnier & Andr Ménez

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  1. Eric Quéméneur
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  2. Mireille Moutiez
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  3. Jean-Baptiste Charbonnier
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Correspondence to Eric Quéméneur.

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Quéméneur, E., Moutiez, M., Charbonnier, JB. et al. Engineering cyclophilin into a proline-specific endopeptidase. Nature 391, 301–304 (1998). https://doi.org/10.1038/34687

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