Characterization of Mycobacterium smegmatis Glutaminase-Free Asparaginase (MSMEG_3173)

l-asparaginase\nis an enzyme catalyzing the hydrolysis\nof l-asparagine into l-aspartate and ammonia, which\nis of great therapeutic importance in tumor treatment. However, commercially\navailable enzymes are associated with adverse effects, and searching\nfor a new l-asparaginase with better pharmaceutical properties\nwas the aim of this work. The coding sequence for Mycobacterium\nsmegmatis l-asparaginase (MsA) was cloned\nand expressed. The recombinant protein showed high activity toward l-asparagine, whereas none was detected for l-glutamine.\nThe enzymatic properties (<i>K</i><sub>m</sub> = 1.403 ±\n0.24 mM and <i>k</i><sub>cat</sub> = 708.1 ± 25.05\ns<sup>–1</sup>) indicate that the enzyme would be functional\nwithin the expected blood l-asparagine concentration, with\ngood activity, as shown by <i>k</i><sub>cat</sub>. The pH\nand temperature profiles suggest its use as a biopharmaceutical in\nhumans. Molecular dynamics analysis of the MsA model reveals the formation\nof a hydrogen bond network involving catalytic residues with l-asparagine. However, the same is not observed with l-glutamine,\nmainly due to steric hindrance. Additionally, the structural feature\nof residue 119 being a serine rather than a proline has significant\nimplications. These findings help explain the low glutaminase activity\nobserved in MsA, like what is described for the Wolinella\nsuccinogenes enzyme. This establishes mycobacterial\nasparaginases as key scaffolds to develop biopharmaceuticals against\nacute lymphocytic leukemia.