Structural Basis for the Activity and Substrate Specificity of Erwinia chrysanthemi l-Asparaginase

Bacterial l-asparaginases, enzymes that catalyze the hydrolysis of l-asparagine to aspartic acid, have been used for over 30 years as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Other substrates of asparaginases include l-glutamine, d-asparagine, and succinic acid monoamide. In this report, we present high-resolution crystal structures of the complexes of Erwinia chrysanthemi l-asparaginase (ErA) with the products of such reactions that also can serve as substrates, namely l-glutamic acid (l-Glu), d-aspartic acid (d-Asp), and succinic acid (Suc). Comparison of the four independent active sites within each complex indicates unique and specific binding of the ligand molecules; the mode of binding is also similar between complexes. The lack of the α-NH3 + group in Suc, compared to l-Asp, does not affect the binding mode. The side chain of l-Glu, larger than that of l-Asp, causes several structural distortions in the ErA active side. The active site flexible loop (residues 15−33) does not exhibit stable conformation, resulting in suboptimal orientation of the nucleophile, Thr15. Additionally, the δ-COO- plane of l-Glu is approximately perpendicular to the plane of γ−COO- in l-Asp bound to the asparaginase active site. Binding of d-Asp to the ErA active site is very distinctive compared to the other ligands, suggesting that the low activity of ErA against d-Asp could be mainly attributed to the low k cat value. A comparison of the amino acid sequence and the crystal structure of ErA with those of other bacterial l-asparaginases shows that the presence of two active-site residues, Glu63ErA and Ser254ErA, may correlate with significant glutaminase activity, while their substitution by Gln and Asn, respectively, may lead to minimal l-glutaminase activity.