Structural Insight into Substrate Selectivity of Erwinia chrysanthemi L-asparaginase

Abstract

l-Asparaginases of bacterial origin are a mainstay of acute lymphoblastic leukemia treatment. The mechanism of action of these enzyme drugs is associated with their capacity to deplete the amino acid l-asparagine from the blood. However, clinical use of bacterial l-asparaginases is complicated by their dual l-asparaginase and l-glutaminase activities. The latter, even though representing only ∼10% of the overall activity, is partially responsible for the observed toxic side effects. Hence, l-asparaginases devoid of l-glutaminase activity hold potential as safer drugs. Understanding the key determinants of l-asparaginase substrate specificity is a prerequisite step toward the development of enzyme variants with reduced toxicity. Here we present crystal structures of the Erwinia chrysanthemi l-asparaginase in complex with l-aspartic acid and with l-glutamic acid. These structures reveal two enzyme conformations-open and closed-corresponding to the inactive and active states, respectively. The binding of ligands induces the positioning of the catalytic Thr15 into its active conformation, which in turn allows for the ordering and closure of the flexible N-terminal loop. Notably, l-aspartic acid is more efficient than l-glutamic acid in inducing the active positioning of Thr15. Structural elements explaining the preference of the enzyme for l-asparagine over l-glutamine are discussed with guidance to the future development of more specific l-asparaginases.

Figure 1

The l-asparaginase and l-glutaminase reactions. The E. chrysanthemil-asparaginase has dual l-asparaginase and l-glutaminase activities. (A) The l-asparaginase reaction. (B) The l-glutaminase reaction.

Figure 2

Binding of ASP versus GLU has no differential effect on the overall Erwinial-asparaginase structure, whereas the ordering of the flexible N-terminal loop is sensitive to the nature of the amino acid ligand. (A) Overlay of the ErA-ASP complex tetramer (each protomer colored differently) and the ErA-GLU complex tetramer (all protomers colored gray) shows an identical overall fold. ASP molecules (purple) from the ErA-ASP complex indicate the location of the four active sites. (B) Overlay of the four individual protomers of the ErA-ASP complex reveals identical protomers with a bound ASP molecule at each active site, and a fully ordered N-terminal loop in all protomers (zoom). (C) Overlay of the four protomers of the ErA-GLU complex (colored as ErA-ASP but using brighter colors) reveals mostly identical protomers with a bound GLU molecule at each active site, but differences in the N-terminal loop. Three protomers (A, B, and C) had no electron density for this flexible loop, whereas for protomer D we could model the loop (zoom). The boundaries of the flexible N-terminal loop are marked with the beginning (18) and ending (33) residue numbers for this region.

Figure 3

ASP binding mode to ErA. (A) Omit density for the ligand ASP in protomer A. (B) Omit density for the Thr15 in protomer A. Map used in A and B is a simulated annealing omit map, contoured at 2.5σ, where the ligand ASP was not included in the model, and Thr15 was mutated to a glycine, for the simulated annealing step (C) Binding of ASP to ErA: polar interactions between ASP and active site residues are indicated with dashed lines, with distances in angstroms. Note the water-mediated interaction between Tyr29, a residue located in the N-terminal loop, and the conserved Thr15.

Figure 4

GLU binding mode to ErA. (A) Omit density for the ligand GLU in protomer A. (B) Omit density for the Thr15 in protomer A. (C) Overlay of the ErA-ASP (pale green) with the ErA-GLU protomer A (bright green). The N-terminal loop was not visible in the ErA-GLU protomer, and Thr15 is displaced and rotated relative to its position in the ErA-ASP structure (gray circle). (D) Omit density for the ligand GLU in protomer D. (E) Omit density for the Thr15 in protomer D. (F) Overlay of the ErA-ASP (pale green) with the ErA-GLU protomer D (orange, open conformation; yellow, closed conformation). We interpret the electron density for the GLU and Thr15 to represent two conformations. At 1/3 occupancy (orange), is the open state as seen in the other protomers of the ErA-GLU structure. At 2/3 occupancy (yellow), is the closed state, which resembles the closed state of the ErA-ASP structure. Map used in (A), (B), (D), and (E) is a simulated annealing omit map, contoured at 2.5σ, where the ligand GLU was not included in the model, and Thr15 was mutated to a glycine, for the simulated annealing step.

Figure 5

Thr15 couples substrate binding and closure of the N-terminal loop. (A) Stereoview showing an overlay of select regions from the ErA-ASP complex (pale green) and the ErA-GLU complex protomers A (bright green) and D (yellow). For the ErA-ASP complex, binding of ligand affects the conformation of Thr15, which in turn promotes closure of the N-terminal loop. In contrast, binding of GLU either did not (protomer A) or only partially (protomer D) induced this change to the Thr15 conformation. Note that the position of most active site residues (Glu63, Thr95, Asp96, and Lys168) is insensitive to the nature of the ligand (ASP versus GLU). In contrast, the conformation of Thr15 is sensitive to the type of ligand. Binding of ASP induces a conformational change in Thr15 that results in a state that is consistent with the closed N-terminal loop. Lacking this conformational change in Thr15, as seen in protomer A of the ErA-GLU structure, the methyl group of the Thr15 side chain would clash with Tyr29. In protomer D of the ErA-GLU structure, binding of GLU did induce a partial rotation of Thr15, which in turn allowed for N-terminal loop closure. (B) Conformation of ASP versus GLU in the Erwinial-asparaginase active site. See text for details.