Engineering cyclophilin into a proline-specific endopeptidase

Area of Science:

• Enzyme engineering
• Protein chemistry
• Biocatalysis

Background:

• Enzyme design necessitates precise catalytic site positioning within substrate-binding pockets.
• Cyclophilins are known for binding proline-containing peptides.

Purpose of the Study:

• To engineer a novel catalytic activity into an Escherichia coli cyclophilin.
• To create a unique endopeptidase with specificity for proline residues.

Main Methods:

• Mutagenesis of three amino acids in an E. coli cyclophilin to mimic serine protease catalytic triads.
• Characterization of the engineered enzyme's (cyproase 1) catalytic efficiency and substrate specificity.
• Assessing inhibition by nucleophile-specific reagents.

Main Results:

• Engineered cyproase 1 exhibits endopeptidase activity, cleaving peptides on the amino-side of proline residues.
• Cyproase 1 demonstrates high catalytic efficiency (kcat/Km = 0.7 x 10^4 M^-1 s^-1) and rate enhancement (kcat/kuncat = 8 x 10^8-fold) on Ala-Pro dipeptide.
• Enzyme activity is dependent on a deprotonated histidine and inhibited by reagents targeting nucleophiles, similar to serine proteases.
• Cyproase 1 successfully hydrolyzes protein substrates with proline-specific endoprotease activity.

Conclusions:

• Successful engineering of a novel proline-specific endopeptidase (cyproase 1) from a cyclophilin scaffold.
• Demonstrates the feasibility of creating new enzymatic activities through targeted mutagenesis and mimicking natural catalytic machinery.
• Highlights the potential for designing bespoke enzymes for specific peptide bond hydrolysis.