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 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.