Two bacterial collagenolytic serine proteases have different topological specificities

Area of Science:

• Biochemistry
• Microbiology
• Enzymology

Background:

• Soil microorganisms, such as Streptomyces omiyaensis, are a rich source of novel enzymes.
• Serine proteases are a diverse class of enzymes with various biotechnological applications.
• Understanding enzyme specificity is crucial for protein engineering and industrial use.

Purpose of the Study:

• To isolate and characterize a novel serine protease (SOT) from Streptomyces omiyaensis.
• To compare the enzymatic properties and substrate specificity of SOT with a related protease, S. griseus trypsin (SGT).
• To identify the protein domain responsible for differences in substrate specificity between SOT and SGT.

Main Methods:

• Isolation and purification of secreted serine protease from Streptomyces omiyaensis.
• Sequence analysis and recombinant protein expression in S. lividans.
• Enzyme characterization including optimum pH, temperature, thermostability, and substrate preference assays.
• Construction and analysis of SOT-SGT chimeras to determine domain function.

Main Results:

• A serine protease (SOT) with high gelatinolytic activity was purified.
• SOT showed 77% sequence identity to SGT but exhibited distinct substrate specificity, particularly towards type IV collagen.
• SOT demonstrated higher hydrolytic activity against gelatin and casein compared to SGT.
• Chimera analysis revealed that the N-terminal domain of SOT is critical for its unique topological specificity.

Conclusions:

• Streptomyces omiyaensis secretes a serine protease (SOT) with potent collagenolytic and gelatinolytic activities.
• Despite high primary structure similarity, SOT and SGT possess different substrate specificities.
• The N-terminal domain plays a key role in determining the topological specificity of these serine proteases.