In Silico-Driven Engineering of Halomonas elongata L-Asparaginase: Towards Enhanced Proteolytic Resistance in Lymphoblastic Leukemia

The shortened L-asparaginases half-life in leukemia patients due to elevated serum proteases, poses a challenge. This study aimed to enhance the stability of Halomonas elongata L-asparaginase against trypsin. Employing the trRosetta server, we modeled the enzymes 3D structure with a quality score of 96.5, revealing predominant secondary structure of random coils (42%), alpha helices (33%), and extended strands (20%) organized in two domains. Molecular docking unveiled a triad alignment among residues Thr16, Ser65, and Asp97 with L-asparagine. Site selection for mutation considered secondary structure prediction, dimerization analysis, trypsin cleavage site determination and epitope mapping. A library of enzyme variants was constructed through site saturation mutagenesis which led to the identification of the Arg206 to Thr, resulting in a 1.7-fold increased enzyme-specific activity (2400 U/mg) and heightened trypsin resistance. The mutant displayed a half-life of 3.47 hin human serum, approximately 50% longer than the wild type. In silico analyses confirmed structural stability, reduced flexibility, and enhanced substrate binding, contributing to increased proteolysis resistance and enzymatic activity. The Arg206Thr mutant exhibited anti-proliferative activity (IC50 of 1.45 U/ml) on leukemia cell line K562, suggesting potential therapeutic implications.