Characterization of Mycobacterium smegmatis Glutaminase-Free Asparaginase (MSMEG_3173)

Abstract

l-asparaginase is an enzyme catalyzing the hydrolysis of l-asparagine into l-aspartate and ammonia, which is of great therapeutic importance in tumor treatment. However, commercially available enzymes are associated with adverse effects, and searching for a new l-asparaginase with better pharmaceutical properties was the aim of this work. The coding sequence for Mycobacterium smegmatisl-asparaginase (MsA) was cloned and expressed. The recombinant protein showed high activity toward l-asparagine, whereas none was detected for l-glutamine. The enzymatic properties (K _m = 1.403 ± 0.24 mM and k _cat = 708.1 ± 25.05 s^-1) indicate that the enzyme would be functional within the expected blood l-asparagine concentration, with good activity, as shown by k _cat. The pH and temperature profiles suggest its use as a biopharmaceutical in humans. Molecular dynamics analysis of the MsA model reveals the formation of a hydrogen bond network involving catalytic residues with l-asparagine. However, the same is not observed with l-glutamine, mainly due to steric hindrance. Additionally, the structural feature of residue 119 being a serine rather than a proline has significant implications. These findings help explain the low glutaminase activity observed in MsA, like what is described for the Wolinella succinogenes enzyme. This establishes mycobacterial asparaginases as key scaffolds to develop biopharmaceuticals against acute lymphocytic leukemia.

Figure 1

Recombinant MsA purification steps. After inducing the proper clone, cells were mechanically disrupted, and total extract (Ext) was clarified by centrifugation, leading to the soluble (Sol) and insoluble (Insol) fractions. The former was used in a Ni²⁺-IMAC, leading to a purified MsA sample in the 20% elution buffer step (A). The latter was further concentrated and buffer exchanged (Amicon). The overall purification was also assessed by analyzing protein complexity in all samples by 12% SDS-PAGE, stained with CBB-R250 (B).

Figure 2

MsA substrate specificity and enzyme kinetics toward l-Asn. (A) Specific activity (U/mg) for MsA and Leuginase (a commercial E. colil-asparaginase 2 preparation) was measured toward l-Asn and l-Gln. Activity using l-Asn as substrate was considered as 100%. (B) Initial velocity (mM/s) using l-Asn as substrate was measured in a range of concentrations (0.05 up to 4 mM). Data were fit in a nonlinear regression, and K_m and k_cat values were calculated by applying the created model. Results are represented as the mean ± the SD of a set of three independent experiments.

Figure 3

Three-dimensional structure of the catalytic sites of MsA and WoA. Pose of l-Asp (represented by the red [oxygen], purple [carbon], and blue [nitrogen] dots) in the MsA site derived from docking assays (A) and the WoA was taken of PDB ID: 5K3O (C). Also represented are mutations S119P in MsA (B) and P121S in WoA (D).

Figure 4

Root-mean-square fluctuation (RMSF) contributed by the first principal component (PC1). Structural representation obtained by displacements along with PC1 is displayed within each plot. Blue shows overlapping regions with little or no motion. Red areas represent mobile regions.

Figure 5

Effect of reaction pH and temperature on MsA asparaginase activity. (A) Asparaginase activity was measured at different pH values. To assess this, several buffers were used (when the buffer salt was exchanged, the same pH value was tested for each composition). (B) Activity was also assessed at different reaction temperatures, at pH 8.6. Values are relative to the higher activity obtained for each assay. Results are represented as the mean ± SD of a set of three independent experiments. (C) Surface electrostatic potential distribution on MsA at different pH values (5.0, 7.0, and 9.0). The surface is color-coded based on the electrostatic potential, with red, white, and blue representing acidic, neutral, and basic potentials, respectively.

Figure 6

Three-dimensional representation of the residue solvent accessibility for each residue of MsA systems. Around the central structure, the regions containing the predicted epitopes of the B cell (A) and CD4+T cell (B) are highlighted. Each residue was colored according to the figure caption.