Controlling enzyme activity by mutagenesis and metal exchange to obtain crystal structures of stable substrate complexes of Class 3 l-asparaginase.

Abstract

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.