Conversion of a regulatory into a degradative protease.

Area of Science:

• Bacterial physiology and molecular biology
• Protein structure-function relationships
• Enzyme kinetics and regulation

Background:

• The sigmaE pathway in bacteria is crucial for responding to unfolded proteins in the periplasm.
• DegS protease initiates this pathway by processing the anti-sigma factor RseA.
• DegS activity is rate-limiting, highly specific, and tightly regulated, but specificity determinants were unknown.

Purpose of the Study:

• To identify the structural determinants responsible for the substrate specificity of the PDZ protease DegS.
• To investigate whether these determinants influence the regulation of DegS activity.

Main Methods:

• Utilized swapping experiments between the homologous proteases DegS and DegP.
• Introduced specific structural elements, including loop L2 and PDZ domains, from DegP into DegS.
• Assessed the substrate specificity and regulatory properties of the modified DegS enzymes.

Main Results:

• Introduction of loop L2 from DegP into DegS converted it into a non-specific protease.
• Swapping of PDZ domains alone did not alter DegS specificity.
• Loop L2 was identified as a key determinant of DegS substrate specificity, without affecting its regulation.
• Combined swapping of loop L2 and PDZ domain 1 further enhanced DegS's DegP-like characteristics.

Conclusions:

• Loop L2 of the protease domain is a critical determinant of substrate specificity for DegS.
• Simple genetic modifications can convert homologous enzymes with distinct activities and regulatory profiles.
• This study provides insights into enzyme engineering and the evolution of protein function.