Thermal Characterization and Interaction of the Subunits from the Multimeric Bacteriophage Endolysin PlyC

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Bacteriophage endolysins are enzymes targeting bacterial peptidoglycan, offering alternatives to antibiotics.
• The multimeric streptococcal endolysin PlyC exhibits potent antibacterial activity.
• Comprehensive thermal stability data for PlyC is essential for assessing its therapeutic viability.

Purpose of the Study:

• To conduct a thorough thermal analysis of the bacteriophage endolysin PlyC and its components.
• To investigate the structural, kinetic, and thermodynamic stability of PlyC under varying conditions.
• To understand the thermal denaturation mechanisms and their impact on PlyC's enzymatic activity.

Main Methods:

• Differential scanning calorimetry (DSC) was employed to assess thermal stability.
• Biochemical and kinetic assays were utilized to evaluate enzyme function and stability.
• Analysis of PlyC holoenzyme, catalytic subunit (PlyCA), and cell wall-binding domain (PlyCB) was performed.

Main Results:

• The PlyC holoenzyme irreversibly denatures and aggregates at 46°C, initiated by PlyCA unfolding, leading to inactivation.
• The PlyCB octamer demonstrates thermostability, with denaturation around 75°C.
• Isolated PlyCA unfolds uncooperatively and is thermodynamically destabilized, while PlyCB reversibly dissociates and denatures at higher temperatures (92°C).
• Active PlyC holoenzyme was successfully reconstituted in vitro from its subunits.

Conclusions:

• PlyC's thermal stability is limited, with denaturation occurring at relatively low temperatures, primarily driven by the PlyCA subunit.
• The PlyCB subunit is significantly more thermostable than PlyCA.
• Understanding these thermal properties is crucial for developing PlyC as a therapeutic agent, with potential for in vitro reconstitution offering a pathway for application.