Crystal structure of the extended-spectrum β-lactamase PER-2 and insights into the role of specific residues in the interaction with β-lactams and β-lactamase inhibitors

Abstract

PER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A ("A" indicates an insertion according to Ambler's scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior.

FIG 1

Amino acid sequence alignment of PER-2 and other class A β-lactamases for which the crystallographic structure has been determined using the Ambler residue numbering. Location of α helices and β sheets is indicated in the upper side (taken from the PDB file), and relative solvent accessibility in the bottom (black, highly accessible; gray, poorly accessible; white, hidden or inaccessible). ESPript (http://espript.ibcp.fr/ESPript/ESPript/) was used to make the figure.

FIG 2

(a) Overall structure of PER-2 β-lactamase, showing the location of the main motifs of the active site (pink), the unique Ω loop (orange), and the three insertions (compared to TEM-1; pale blue). (b) Detail of the four-residue insertion in PER-2 (pink) that creates an expanded loop between β3 and β4 strands, widening the active site entrance (orange, TEM-1; green, TOHO-1). (c) Comparison between the singular trans bond between Glu166-Ala167 and hydrogen bonds with Asp136 in PER-2 (pink) and the normally cis bond between Glu166-Pro167 (and hydrogen bonds with Asn136) found in other class A β-lactamases like TOHO-1 (green). All distances are in angstroms (Å).

FIG 3

Detailed view of the structure of active site of PER-2 β-lactamase. (a) 2FoFc map contoured at 1.5σ is shown in gray around the most important amino acid residues within the active site; oxyanion water molecule is shown as a green sphere, and additional water molecules in orange (see the Results and Discussion for details). (b) Comparative active site organization of PER-2 (pink) and PER-1 (cyan), indicating the main hydrogen bonds (black dashed lines) implicated in the stabilization of the active site of PER-2, including the oxyanion water molecules (OAW) (green for PER-2 and orange for PER-1) and the catalytic water of PER-1 (CW) (orange), and the network Ser70-Gln69-Wat14-Thr237-Arg220 (see Results and Discussion for details); for visual convenience, only the hydrogen bonds for PER-2 were shown. (c) Position and occupancy of Arg220 in PER-2, allowing the creation of a unique network of hydrogen bonds with neighboring residues like Gly236, Thr237, Asn245, and Glu276, among others; Ser70 is shown as reference. Other color references: red, oxygen; blue, nitrogen; green, sulfur. All distances are in angstroms (Å).

FIG 4

(a) Detailed view of the active site of TOHO-1 (aqua) in association with cefotaxime (yellow) (left), indicating the main hydrogen bond interactions (PDB 1IYO), and simulated modeling of PER-2 (pink) and the probable positioning of cefotaxime within the active site (right), suggesting the putative most favorable hydrogen bonds and involvement of residues like Gln69, Thr237, and Arg240A in the stabilization of the oxyimino-cephalosporin molecule. (b) Active site of TOHO-1 in complex with acylated ceftazidime (magenta) (left), indicating the main hydrogen bonds (PDB 2ZQD), compared to a simulated model of PER-2 and its probable association with ceftazidime (right), showing the predicted positioning of the molecule and the hydrogen bond interactions (black dashed lines) All distances are in angstroms (Å). Other color references: red, oxygen; blue, nitrogen; green, sulfur. See Results and Discussion for details.