title: Engineering cyclophilin into a proline-specific endopeptidase.
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 complexes, 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 x 10(4) M(-1) s(-1) and enhances the rate of reaction (kcat/kuncat) 8 x 10(8)-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.
authors.affiliation: Département d'Ingénierie et d'Etudes des Protéines, CEA Saclay, Gif-sur-Yvette, France. eric.quemeneur@cea.fr
authors.forename: E
authors.initials: E
authors.lastname: Quéméneur
authors.forename: M
authors.initials: M
authors.lastname: Moutiez
authors.forename: J B
authors.initials: JB
authors.lastname: Charbonnier
authors.forename: A
authors.initials: A
authors.lastname: Ménez
mesh_terms: D002384:Catalysis; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008968:Molecular Conformation; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism*; D000085822:Prolyl Oligopeptidases; D015202:Protein Engineering; D012694:Serine / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D012694:Serine; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; C109933:cyproase 1; D019696:Peptidylprolyl Isomerase