Engineering cyclophilin into a proline-specific endopeptidase.
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.
Quéméneur
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Département d'Ingénierie et d'Etudes des Protéines, CEA Saclay, Gif-sur-Yvette, France. eric.quemeneur@cea.fr
Moutiez
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M
Charbonnier
J B
JB
Ménez
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A
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
D012694:Serine; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; C109933:cyproase 1; D019696:Peptidylprolyl Isomerase