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 (Tm), 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 Km 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 (Km 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.

Common bean ( Phaseolus vulgaris ) encodes three class 2 L-asparaginase enzymes: two potassium-dependent enzymes [PvAIII(K)-1 and PvAIII(K)-2] and a potassium-independent enzyme (PvAIII). Here, we present the crystal structure of PvAIII, which displays a rare P 2 space-group symmetry and a unique pseudosymmetric 4 1 -like double-helical packing. The asymmetric unit contains 32 protein chains (16 αβ units labeled A – P ) organized into two right-handed coiled arrangements, each consisting of four PvAIII (αβ) 2 dimers. Detailed analysis of the crystal structure revealed that this unusual packing originates from three factors: (i) the ability of the PvAIII molecules to form extended intermolecular β-sheets, a feature enabled by the PvAIII sequence and secondary structure, (ii) incomplete degradation of the flexible linker remaining at the C-terminus of α subunits of protein chain C after the autoproteolytic cleavage (maturation) of the PvAIII precursor and (iii) intermolecular entanglement between protein chains from the two helices to create `hydrogen-bond linchpins' that connect adjacent protein chains. The K m value of PvAIII for L-asparagine is approximately five times higher than for β-peptides, suggesting that the physiological role of PvAIII may be more related to the removal of toxic β-peptides than to basic L-asparagine metabolism. A comparison of the active sites of PvAIII and PvAIII(K)-1 shows that the proteins have nearly identical residues in the catalytic center, except for Thr219, which is unique to PvAIII. To test whether the residue type at position 219 affects the enzymatic activity of PvAIII, we designed and produced a T219S mutant. The kinetic parameters determined for L-asparagine hydrolysis indicate that the T/S residue type at position 219 does not affect the L-asparaginase activity of PvAIII.

The ReAV enzyme from Rhizobium etli, a representative of Class 3 L-asparaginases, is sequentially and structurally different from other known L-asparaginases. This distinctiveness makes ReAV a candidate for novel antileukemic therapies. ReAV is a homodimeric protein, with each subunit containing a highly specific zinc-binding site created by two cysteines, a lysine, and a water molecule. Two Ser-Lys tandems (Ser48-Lys51, Ser80-Lys263) are located in the close proximity of the metal binding site, with Ser48 hypothesized to be the catalytic nucleophile. To further investigate the catalytic process of ReAV, site-directed mutagenesis was employed to introduce alanine substitutions at residues from the Ser-Lys tandems and at Arg47, located near the Ser48-Lys51 tandem. These mutational studies, along with enzymatic assays and X-ray structure determinations, demonstrated that substitution of each of these highly conserved residues abolished the catalytic activity, confirming their essential role in enzyme mechanism.

The Philadelphia positive acute lymphoblastic leukemia (ALL BCR::ABL1 positive) is characterized by the presence of the t(9;22)(q34;q11) resulting in the formation of the BCR::ABL1 fusion, constant high cellular BCR-ABL1 tyrosine kinase activity, abnormal lymphoid cell proliferation and genetic instability of leukemic cells. Recently, great progress has been made due to the incorporation of tyrosine kinase inhibitors (TKI’s) into the treatment algorithms. Despite this, a significant number of patients experienced therapy resistance mainly due to disease evolution and/or acquisition of TKI resistant BCR::ABL1 mutation(s). Herein, we report the therapy outcome in a 65 year old woman with high-risk BCR::ABL1 positive ALL treated with the PALG ALL7 (including imatinib, IM) protocol, who obtained complete hematologic remission (CHR) and complete molecular remission (CMR) after treatment with induction,consolidation I and II. Despite the fully matched donor identification, the patient refused to continue with intensive chemotherapy. Therefore, only IM therapy was continued, with periodic drug dose reduction due to hematological toxicity. CHR loss with the absence of BCR::ABL1 KD mutation was noticed 10 months later. The treatment with dasatinib (DAS) 140mg/d and dexamethasone resulted in a second CMR. Due to gastrointestinal toxicity, the DAS dose was reduced to 100mg/d. The second disease relapse was diagnosed 3 months later. BCR::ABL1 KD mutation screening showed the Lys404Glu (K404E) substitution. The 3-week long treatment with ponatinib in the dose of 45mg/d resulted in disease progression. The reinduction therapy with anti-CD22 MoAb (Inotuzumab ozogamycin) led to the 3rd CMR (duration time 9 months). According to our knowledge, it is the first report on the emergence of a BCR::ABL1 Lys404Glu mutation in an ALL patient receiving TKI treatment. Due to the mutation emergence on DAS, and disease progression on subsequent ponatinib treatment, the impact of the mutation acquisition on the 3D structure of ABL1 molecule and the process of TKI’s binding to ATP-binding site were additionally studied. The results of the study indicate that the TKI resistance observed in the presented case is probably the result of the impossibility of the formation of stable complexes between ATP-competitive inhibitors and mutant Lys404Glu BCR-ABL1 tyrosine kinase due to conformational molecule changes.

Rhizobium etli is a nitrogen-fixing bacterium that encodes two l-asparaginases. The structure of the inducible R. etli asparaginase ReAV has been recently determined to reveal a protein with no similarity to known enzymes with l-asparaginase activity, but showing a curious resemblance to glutaminases and β-lactamases. The uniqueness of the ReAV sequence and 3D structure make the enzyme an interesting candidate as potential replacement for the immunogenic bacterial-type asparaginases that are currently in use for the treatment of acute lymphoblastic leukemia. The detailed catalytic mechanism of ReAV is still unknown; therefore, the enzyme was subjected to mutagenetic experiments to investigate its catalytic apparatus. In this work, we generated two ReAV variants of the conserved Lys138 residue (K138A and K138H) that is involved in zinc coordination in the wild-type protein and studied them kinetically and structurally. We established that the activity of wild-type ReAV and the generated variants is significantly reduced in the presence of Cd2+ cations, which slow down the proteins while improving their apparent substrate affinity. Moreover, the inhibitory effect of Cd2+ is enhanced by the substitutions of Lys138, which disrupt the metal coordination sphere. The proteins with impaired activity but increased affinity were cocrystallized with the L-Asn substrate. Here, we present the crystal structures of wild-type ReAV and its K138A and K138H variants, unambiguously revealing bound l-asparagine in the active site. After careful analysis of the stereochemistry of the nucleophilic attack, we assign the role of the primary nucleophile of ReAV to Ser48. Furthermore, we propose that the reaction catalyzed by ReAV proceeds according to a double-displacement mechanism.

L-asparaginases are used in the treatment of acute lymphoblastic leukemia. The aim of this work was to compare the antiproliferative potential and proapoptotic properties of novel L-asparaginases from different structural classes, viz. EcAIII and KpAIII (class 2), as well as ReAIV and ReAV (class 3). The EcAII (class 1) enzyme served as a reference. The proapoptotic and antiproliferative effects were tested using four human leukemia cell models: MOLT-4, RAJI, THP-1, and HL-60. The antiproliferative assay with the MOLT-4 cell line indicated the inhibitory properties of all tested L-asparaginases. The results from the THP-1 cell models showed a similar antiproliferative effect in the presence of EcAII, EcAIII, and KpAIII. In the case of HL-60 cells, the inhibition of proliferation was observed in the presence of EcAII and KpAIII, whereas the proliferation of RAJI cells was inhibited only by EcAII. The results of the proapoptotic assays showed individual effects of the enzymes toward specific cell lines, suggesting a selective (time-dependent and dose-dependent) action of the tested L-asparaginases. We have, thus, demonstrated that novel L-asparaginases, with a lower substrate affinity than EcAII, also exhibit significant antileukemic properties in vitro, which makes them interesting new drug candidates for the treatment of hematological malignancies. For all enzymes, the kinetic parameters (Km and kcat) and thermal stability (Tm) were determined. Structural and catalytic properties of L-asparaginases from different classes are also summarized.

This report describes a comprehensive approach to local random mutagenesis of the E. coli Ntn-amidohydrolase EcAIII, and supplements the results published earlier for the randomization series RDM1. Here, random mutagenesis was applied in the center of the EcAIII molecule, i.e., in the region important for substrate binding and its immediate neighborhood (series RDM2, RDM3, RDM7), in the vicinity of the catalytic threonine triplet (series RDM4, RDM5, RDM6), in the linker region (series RDM8), and in the sodium-binding (stabilization) loop (series RDM9). The results revealed that the majority of the new EcAIII variants have abolished or significantly reduced rate of autoprocessing, even if the mutation was not in a highly conserved sequence and structure regions. AlphaFold-predicted structures of the mutants suggest the role of selected residues in the positioning of the linker and stabilization of the scissile bond in precisely correct orientation, enabling the nucleophilic attack during the maturation process. The presented data highlight the details of EcAIII geometry that are important for the autoproteolytic maturation and for the catalytic mechanism in general, and can be treated as a guide for protein engineering experiments with other Ntn-hydrolases.

The absence of solvent molecules in high-resolution protein crystal structure models deposited in the Protein Data Bank (PDB) contradicts the fact that, for proteins crystallized from aqueous media, water molecules are always expected to bind to the protein surface, as well as to some sites in the protein interior. An analysis of the contents of the PDB indicated that the expected ratio of the number of water molecules to the number of amino-acid residues exceeds 1.5 in atomic resolution structures, decreasing to 0.25 at around 2.5 Å resolution. Nevertheless, almost 800 protein crystal structures determined at a resolution of 2.5 Å or higher are found in the current release of the PDB without any water molecules, whereas some other depositions have unusually low or high occupancies of modeled solvent. Detailed analysis of these depositions revealed that the lack of solvent molecules might be an indication of problems with either the diffraction data, the refinement protocol, the deposition process or a combination of these factors. It is postulated that problems with solvent structure should be flagged by the PDB and addressed by the depositors.

L-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low sequence identity. In the present work we examined the biochemical consequences of this difference. ReAIV is almost twice as efficient as ReAV in asparagine hydrolysis at 37°C, with the kinetic KM, kcat parameters (measured in optimal buffering agent) of 1.5 mM, 770 s-1 and 2.1 mM, 603 s-1, respectively. The activity of ReAIV has a temperature optimum at 45°C–55°C, whereas the activity of ReAV, after reaching its optimum at 37°C, decreases dramatically at 45°C. The activity of both isoforms is boosted by 32 or 56%, by low and optimal concentration of zinc, which is bound three times more strongly by ReAIV then by ReAV, as reflected by the KD values of 1.2 and 3.3 μM, respectively. We also demonstrate that perturbation of zinc binding by Lys→Ala point mutagenesis drastically decreases the enzyme activity but also changes the mode of response to zinc. We also examined the impact of different divalent cations on the activity, kinetics, and stability of both isoforms. It appeared that Ni2+, Cu2+, Hg2+, and Cd2+ have the potential to inhibit both isoforms in the following order (from the strongest to weakest inhibitors) Hg2+ > Cu2+ > Cd2+ > Ni2+. ReAIV is more sensitive to Cu2+ and Cd2+, while ReAV is more sensitive to Hg2+ and Ni2+, as revealed by IC50 values, melting scans, and influence on substrate specificity. Low concentration of Cd2+ improves substrate specificity of both isoforms, suggesting its role in substrate recognition. The same observation was made for Hg2+ in the case of ReAIV. The activity of the ReAV isoform is less sensitive to Cl− anions, as reflected by the IC50 value for NaCl, which is eightfold higher for ReAV relative to ReAIV. The uncovered complementary properties of the two isoforms help us better understand the inducibility of the ReAV enzyme.