Engineering methionine γ-lyase from Citrobacter freundii for anticancer activity

Area of Science:

• Biochemistry
• Enzymology
• Cancer Therapeutics

Background:

• Cancer cells often lack methionine biosynthesis, making them vulnerable to methionine deprivation.
• Methionine gamma-lyase (MGL) degrades methionine, offering a strategy to reduce its availability to cancer cells.

Purpose of the Study:

• To enhance the catalytic activity of MGL for improved cancer therapy.
• To investigate the role of the C-terminal flexible loop in MGL activity and allosteric regulation.

Main Methods:

• Sequence and structure conservation analysis of MGLs.
• Site-saturation mutagenesis of the C-terminal flexible loop (P357, V358, P360, A366) in Citrobacter freundii MGL.
• Screening of mutant libraries for enzyme activity and cytotoxic effects.
• 3D-structure comparison of wild-type and mutant MGLs.

Main Results:

• Mutagenesis identified active variants, including V358Y MGL with a 1.9-fold increased catalytic rate.
• V358Y MGL exhibited lower IC50 values against cancer cell lines compared to wild-type MGL.
• Structural analysis revealed C-terminal loop flexibility but indicated mutations allosterically affect catalysis.

Conclusions:

• Engineering MGL, particularly at the V358 position, can enhance its cytotoxic potential against cancer cells.
• The C-terminal flexible loop plays a crucial role in MGL's allosteric regulation and catalytic efficiency.
• Modified MGL holds promise as a targeted cancer therapeutic strategy by exploiting methionine dependency.