Plant-derived L-asparaginases: Comprehensive functional and biological characterization

In this study, K-dependent and K-independent plant-derived type III L-asparaginases from Arabidopsis thaliana (AtAIII, AtAIII(K)) and Phaseolus vulgaris (PvAIII, PvAIII(K)-1) were systematically tested for their catalytic properties, structural stability (T_m), and antiproliferative and proapoptotic activity in human leukemia cell lines. Among them, the K-dependent enzyme PvAIII(K)-1 showed the most favorable biological profile, combining high selectivity for L-Asn-dependent MOLT-4 leukemia cells with minimal effects on non-ALL cell lines (RAJI, HL-60, THP-1) and healthy lymphocytes, confirming lack of L-glutaminase co-activity. Although PvAIII(K)-1 exhibits a K_m for L-Asn hydrolysis in the millimolar range (4.13 ± 1.29 mM), consistent with other plant-derived L-asparaginases, exposure of MOLT-4 cells to PvAIII(K)-1 resulted in antiproliferative and proapoptotic effects within 24 h, with an IC₅₀ of 0.0056 mg/mL. Importantly, the detectable β-aspartyl peptidase co-activity (K_m 2.26 ± 0.26 mM), does not appear to compromise biological activity. NanoDSF showed that the K-independent enzymes AtAIII (76.26 °C) and PvAIII (67.22 °C) exhibit higher thermal stability than the K-dependent variants PvAIII(K)-1 (50.57 °C) and AtAIII(K) (45.45 °C). Site-directed mutagenesis of PvAIII(K)-1 revealed that Glu81 and Arg311 are important for protein thermal stability. Molecular docking, and MD simulations indicated that AtAIII exhibits reduced substrate specificity compared to AtAIII(K), PvAIII, and PvAIII(K)-1 due to presence of Tyr204 in the active site, which disrupts the positioning of Arg211 essential for stable L-Asn binding. The results highlight key structural and functional features of plant-derived enzymes that may guide their further rational engineering and optimization.