Genome mining and computational prioritization of a promising L-Asparaginase candidate from Pseudomonas asiatica strain PNPG3

L-asparaginase (Asp) is a biotechnologically important enzyme with diverse applications, particularly in the treatment of acute lymphoblastic leukemia (ALL) and certain solid tumors, as well as in reducing acrylamide formation during food processing. Currently, Asp isoforms derived from Erwinia chrysanthemi and Escherichia coli ( E. coli ) are clinically approved and commercially used forms, owing to their ability to deplete extracellular L-asparagine, which is required for the proliferation of certain neoplastic cells. Genome analysis of Pseudomonas asiatica strain PNPG3 identified three putative Asp-encoding genes. Asp1 and Asp2 showed 35.74% and 33.12% sequence identity to the L-asparaginases of E. coli K-12 and Streptococcus pneumoniae , respectively, based on comparisons with structures available in the PDB database. The relatively low sequence identity indicates that these enzymes may represent previously uncharacterized members of the Asp family. Among the identified enzymes, Asp2 exhibited docking interactions with L-asparagine, forming multiple hydrogen bonds along with significant hydrophobic interactions within the active site. Asp2-L-asparagine complex exhibited comparatively lower structural fluctuations during the simulations, as evidenced by consistently low RMSD values with an average of approximately 0.414 ± 0.11 nm. Furthermore, the complex maintained low and relatively uniform RMSF, Rg profiles, along with stable SASA values and compact PCA clustering, indicating a relatively compact structural conformation with minimal solvent exposure. MM/PBSA interaction energy analysis revealed a ΔG_binding of − 18.05 ± 3.68 kcal/mol for the above complex. In comparison, the positive control exhibited weaker binding (-1.96 ± 1.58 kcal/mol), indicating a more stable and favorable interaction for Asp2-L-asparagine. Overall, these findings suggest that strain PNPG3 may represent a promising source of a computationally prioritized lead enzyme, a conclusion further supported by the halo-based qualitative confirmation of Asp activity. Collectively, these findings indicate that PNPG3 warrants further investigation as a potential source of Asp and provides valuable insights that may inform future strategies for optimizing enzyme production.