Directed Mutagenesis of Catalytic Residues in L-Asparaginase II from Salmonella paratyphi: Structural, Functional and Stability Analysis Using In-Vitro, Docking and Simulation Studies.

L-asparaginase is a clinically crucial enzyme widely used in the treatment of acute lymphoblastic leukemia. However, its therapeutic efficacy is often limited by insufficient stability and activity. In this study, site-directed mutagenesis was employed to modify key residues in L-asparaginase from Salmonella Paratyphi, aiming to enhance both catalytic and structural stability. So, we employed site directed mutagenesis to modify recSalA, resulting in three variants: D103V, (Q81L+ D103V), and Q81L. Molecular docking and simulations predicted that the D103V mutant of Salmonella paratyphi L-asparaginase II exhibited the highest substrate affinity (-6.18 kcal/mol), attributed to enhanced hydrophobic packing and stable hydrogen bonding with key catalytic residues. Throughout the 100 ns molecular dynamics simulation, the (D103V-L-ASN) complex maintained structural stability, with consistent RMSD (~ 2.3 Å) and hydrogen-bond interactions exceeding 70% occupancy. Moreover, it exhibited the highest specificity and L-ASN activity (229.5 U/mg), surpassing even that of the wild type L-asparaginase. All mutant enzymes exhibited peak activity at 40 °C, similar to the native enzyme. Notably, the D103V variant exhibited enhanced stability, with an in vitro activity half-life of 1 h and 27 min, nearly twice the thermal stability of the wild-type enzyme at 37 °C. Furthermore, complementary secondary-structure analyses using PSIPRED and DSSP revealed a highly conserved structural organization, with comparable proportions of α-helices, β-strands, and coil/loop regions across all recSalA variants. These findings indicate that the introduced mutations preserve the overall protein architecture, suggesting that the observed differences in enzymatic activity arise primarily from localized modifications in residue interactions and the catalytic/substrate-binding microenvironment rather than from global conformational changes. Overall, these findings demonstrate that rational site-directed mutagenesis can effectively enhance both the catalytic efficiency and thermal stability of recSalA. Among the engineered variants, D103V and the double mutant (Q81L+D103V) exhibited the most favorable functional properties, highlighting their potential as promising candidates for therapeutic applications. These results also provide valuable structural and functional insights that may facilitate the rational design of more stable and efficacious enzyme formulations.