Computational Insights into Cyst(e)inase for Cysteine and Cystine Depletion Therapies: Free Energy Calculations, Evolutionary Conservation, and Molecular Dynamics Simulations

Cyst(e)inase, an engineered human cystathionine-γ-lyase (hCGL), effectively degrades l-Cys and CSSC, inhibiting various tumors in vivo with minimal toxicity. However, its current activity and stability limit its clinical application. To enhance its therapeutic potential, we utilized folding free energy calculations and amino acid site conservation to identify 46 potential mutants. Among them, 17 mutants exhibited improved activity or stability, while six showed a loss of function. Notably, mutant L142E enhanced activity for both substrates, and E349S displayed 2.8 times higher activity toward CSSC and a 12.8 °C increase in <i>T</i>_m, despite reduced activity toward l-Cys. Saturation mutagenesis highlighted the importance of spatial constraints for substrate selectivity. MD simulations suggested that distal mutations stabilize the near-attack conformation, enhancing the activity. In vitro cytotoxicity assays confirmed that the mutants L142E and E349S significantly inhibited gastric and pancreatic cancer cells. This study provides valuable insights for improving therapeutic enzyme design.