Authors
Mengyi Zhang, Mingyue Liu, Yunlan Tang, Junmeng Li, Chaojie Wang, Weifeng Zhao, Yao Cui
Published in
European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. Pages 107705. Nov 03, 2026. Epub Nov 03, 2026.
Abstract
Arginine deiminase (ADI) is a promising therapeutic enzyme for targeting arginine-auxotrophic cancers, but its clinical application is limited by insufficient catalytic efficiency and poor stability. In this study, we developed a rational protein engineering strategy integrating folding free energy (ΔΔG) prediction and evolutionary conservation analysis to optimize ADI from Pseudomonas mendocina. A comprehensive site-saturation mutational scan was performed using the Pythia framework, followed by ConSurf-based filtering to exclude highly conserved residues, thereby efficiently identifying candidate beneficial mutations. Experimental evaluation showed that several single mutations enhanced enzymatic activity, and combinatorial optimization further revealed synergistic effects. Notably, the triple mutant K91L/N95E/Y346E exhibited the most significant improvement, achieving a 2.7-fold increase in catalytic efficiency (kcat/KM) compared to the wild type. In addition, this variant demonstrated enhanced thermal stability and improved plasma stability, with an extended half-life and higher residual activity. Importantly, in vitro cytotoxicity assays confirmed that the engineered mutant displayed superior antitumor activity against colorectal cancer cells. Overall, this study highlights the effectiveness of integrating thermodynamic and evolutionary constraints for enzyme engineering and provides a promising strategy for developing ADI-based anticancer therapeutics.
PMID:
42977043
Bibliographic data and abstract were imported from PubMed on 04 Nov 2026.
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