Molecular dissection of Streptomyces trypsin on substrate recognition

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Serine proteases SOT and SGT from Streptomyces species exhibit distinct topological specificities.
• Residue 71 was previously identified as crucial for these specificity differences.

Purpose of the Study:

• To investigate the role of residue 71 in substrate recognition and collagen binding.
• To identify other residues involved in collagen interaction.

Main Methods:

• Surface plasmon resonance analysis of inactive serine protease mutants.
• In vivo DNA shuffling of Streptomyces omiyaensis (SOT) and Streptomyces griseus (SGT) proteases.
• Site-directed mutagenesis of identified residues.

Main Results:

• Residue 71 plays a role in substrate specificity and collagen binding affinity.
• Residue 72 was identified as another key residue contributing to collagen binding.
• A mutant SGT with substitutions at residues 71 and 72 exhibited altered specificity and high activity toward type IV collagen, similar to SOT.

Conclusions:

• Neighboring residues 71 and 72 in the N-terminal β-barrel domain synergistically determine substrate recognition and collagen specificity.
• Enzyme engineering by modifying these residues can alter protease function and substrate preference.