A potential antitumor drug (arginine deiminase) reengineered for efficient operation under physiological conditions

Area of Science:

• Biochemistry
• Enzyme Engineering
• Cancer Therapeutics

Background:

• Arginine deiminase (ADI) is a promising antitumor agent for arginine-auxotrophic cancers like HCC and melanoma.
• Existing ADI enzymes, such as Pseudomonas plecoglossicida ADI (PpADI), exhibit pH-dependent activity, with significant loss of function at physiological pH (7.35–7.45).
• Previous engineering efforts yielded variant M2 with improved pH optimum and catalytic efficiency.

Purpose of the Study:

• To engineer ADI variants with optimal activity at physiological pH (7.4) for enhanced therapeutic potential.
• To improve the catalytic efficiency and substrate affinity of ADI for arginine.

Main Methods:

• Directed evolution was employed using Phosphate-Buffered Saline (PBS) buffer at pH 7.4 to mimic physiological conditions.
• Mutagenesis and screening were performed to identify variants with improved enzyme kinetics.
• Kinetic parameters, including S(0.5) and k(cat), were determined for wild-type and engineered ADI variants.

Main Results:

• Two novel ADI variants, M5 and M6, were generated with significantly improved performance at pH 7.4.
• Variant M6 exhibited a lower S(0.5) value (0.81 mM) compared to wild-type PpADI (1.30 mM), indicating enhanced substrate affinity.
• Catalytic efficiency (k(cat)) was substantially increased: M5 showed a 97.6-fold improvement (17.56 s⁻¹) and M6 a 64.7-fold improvement (11.64 s⁻¹) over wild-type PpADI (0.18 s⁻¹).

Conclusions:

• Engineered ADI variants M5 and M6 demonstrate superior enzymatic activity and affinity at physiological pH.
• These variants represent promising candidates for developing more effective arginine deiminase-based cancer therapies.
• The directed evolution approach is effective for optimizing enzyme function under specific physiological conditions.