Crystal structure of the carbapenemase OXA-24 reveals insights into the mechanism of carbapenem hydrolysis

Abstract

Combating bacterial resistance to beta-lactams, the most widely used antibiotics, is an emergent and clinically important challenge. OXA-24 is a class D beta-lactamase isolated from a multiresistant epidemic clinical strain of Acinetobacter baumannii. We have investigated how OXA-24 specifically hydrolyzes the last resort carbapenem antibiotic, and we have determined the crystal structure of OXA-24 at a resolution of 2.5 A. The structure shows that the carbapenem's substrate specificity is determined by a hydrophobic barrier that is established through the specific arrangement of the Tyr-112 and Met-223 side chains, which define a tunnel-like entrance to the active site. The importance of these residues was further confirmed by mutagenesis studies. Biochemical and microbiological analyses of specific point mutants selected on the basis of structural criteria significantly reduced the catalytic efficiency (k(cat)/K(m)) against carbapenems, whereas the specificity for oxacillin was noticeably increased. This is the previously unrecognized crystal structure that has been obtained for a class D carbapenemase enzyme. Accordingly, this information may help to improve the development of effective new drugs to combat beta-lactam resistance. More specifically, it may help to overcome carbapenem resistance in A. baumannii, probably one of the most worrying infectious threats in hospitals worldwide.

Fig. 1.

Structure of OXA-24, a class D carbapenem-hydrolyzing oxacillinase. (a) Stereodiagram showing the overall folding of OXA-24 with the two domains colored differently: the α helical domain is shown in navy blue and in the mixed α/β domain, the β-sheet is shown in cyan, and the α-helices are shown in orange. (b) Surface potential of the tunnel-like entrance to the active site, where blue is positive and red is negative. The sulfate ion and water molecules are shown in ball-and-stick mode.

Fig. 2.

Details of the structure of OXA-24. (a and b) Selected views of the superposition of OXA-24 (cyan) and the oxacillinase members of class D β-lactamases of known structure, OXA-1 (yellow), OXA-2 (navy blue), OXA-10 (red), and OXA-13 (green), highlighting the major structural differences. OXA-24 displays an elongated α-helix (α1) at the N terminus and the β3 strand implicated in dimerization in other oxacillinases is directed toward the C-terminal helix (α9). Important differences can also be observed in the Ω-loop. (c) Detail of the nonpolar region at the lower border of the active binding cleft. The hydrophobic core formed by several nonpolar residues (Val-78, Val-225, Val-229, and Trp-231) contributes to the stability of the solvent-exposed β4-β5 loop and partially blocks the access to the bottom part of the cleft.

Fig. 3.

Close-up view of the β-lactam active site in OXA-24. (a) Representation of the active site elements (17). The first element (gray) is located at the N-terminal end of the 3₁₀-helix α3, and it is formed by residues Ser-81-Thr-82-Phe-83-Lys-84. The second motif (green), Ser-128-Ala-129-Val-130, lies in the loop connecting helices α4 and α5. The third active site element (cyan) is formed by the residues Lys-218-Ser-219-Gly-220 from the β4 strand. (b) Detailed interactions showing the contact network at the active site (shown as solid lines).

Fig. 4.

Acquisition of carbapenem specificity in OXA-24. (a and b) Surface potential drawing in two different orientations of OXA-24 (Left) and OXA-10 (Right) with positive and negative-charged regions shown in blue and red, respectively. Residues Tyr-112 and Met-223 and their structural equivalents in OXA-10, Met-99, and Gly-210 are colored green. The relative orientation of both key residues in OXA-24 (Left) specifically delimits access to the catalytic binding site, whereas the cleft is fully open in OXA-10. (c) Chemical structures of indicated antibiotics.

Fig. 5.

The effect of point mutations on substrate selectivity. (a and b) Docking of imipenem (a) and meropenem (b) within the active site. The substrate is visibly anchored into the tunnel-like cavity of the binding site. Models of OXA-24 with carbapenems, based on the published structures (18), were manually fitted onto the active site and then subjected to energy minimization by using the CNS program (16). (c) Schematic representation of the active site of OXA-24 with a hypothetical oxacillin molecule. The bulk side-chain of this β-lactam antibiotic, at position 6 of the β-lactam ring, collides with the hydrophobic barrier at the tunnel-like entrance. (d) Important catalytic residues in the active site. The Tyr-112 residue seems to be essential for catalytic efficiency against carbapenems because it hooks the 6-α-hydroxyethyl group of these β-lactam antibiotics before catalysis. (e) The hypothetical active site cavity of the OXA-24 double mutant. The binding modus of oxacillin in the double mutant mimics the specific binding of isoxazolyl penicillin derivatives to the open cleft of oxacillinases (17, 19).