Abstract: Cystathionine γ-lyase (CGL) mutant cyst(e)inase effectively degrades cystine (CSSC) and cysteine (L-Cys), exhibiting antitumor activity through redox homeostasis disruption in tumor cells. However, its efficacy has not yet met the requirements for clinical application. To enhance the catalytic activity and thermodynamic stability of cyst(e)inase for CSSC and L-Cys degradation, this study focused on evolutionarily conserved amino acid residues using virtual site-saturation mutagenesis coupled with free energy calculations to identify potential beneficial mutants. The screening of 119 amino acid sites identified 32 promising mutants through free energy analysis. Experimental validation revealed that 8 mutants exhibited enhanced activity toward CSSC and L-Cys. Among these, the top-performing mutant, S206I, demonstrated 40% and 60% higher activity against CSSC and L-Cys, respectively, accompanied by 23% and 230% k_cat/K_m improvements. Additionally, its thermal stability (T_m) was elevated by 1.89 ℃. This study employed an efficient and straightforward design strategy, produced enhanced mutants with enhanced activity and stability, establishing a framework for subsequent therapeutic enzyme engineering.

Key words: cystathion γ-lyase, rational design, free energy calculation, sequence conservatism, enzyme kinetics