Clinical Variants of the Native Class D ß-Lactamase of Acinetobacter baumannii Pose an Emerging Threat through Increased Hydrolytic Activity against Carbapenems.

The threat posed by the chromosomally encoded class D ß-lactamase of Acinetobacter baumannii (OXA-51/66) has been unclear, in part because of its relatively low affinity and turnover rate for carbapenems. Several hundred clinical variants of OXA-51/66 have been reported, many with substitutions of active-site residues. We determined the kinetic properties of OXA-66 and five clinical variants with respect to a wide variety of ß-lactam substrates. The five variants displayed enhanced activity against carbapenems and in some cases against penicillins, late-generation cephalosporins, and the monobactam aztreonam. Molecular dynamics simulations show that in OXA-66, P130 inhibits the side-chain rotation of I129 and thereby prevents doripenem binding because of steric clash. A single amino acid substitution at this position (P130Q) in the variant OXA-109 greatly enhances the mobility of both I129 and a key active-site tryptophan (W222), thereby facilitating carbapenem binding. This expansion of substrate specificity represents a very worrisome development for the efficacy of ß-lactams against this troublesome pathogen.

The structure of a doripenem-bound OXA-51 class D ß-lactamase variant with enhanced carbapenemase activity.

OXA-51 is a class D ß-lactamase that is thought to be the native carbapenemase of Acinetobacter baumannii. Many variants of OXA-51 containing active site substitutions have been identified from A. baumannii isolates, and some of these substitutions increase hydrolytic activity toward carbapenem antibiotics. We have determined the high-resolution structures of apo OXA-51 and OXA-51 with one such substitution (I129L) with the carbapenem doripenem trapped in the active site as an acyl-intermediate. The structure shows that acyl-doripenem adopts an orientation very similar to carbapenem ligands observed in the active site of OXA-24/40 (doripenem) and OXA-23 (meropenem). In the OXA-51 variant/doripenem complex, the indole ring of W222 is oriented away from the doripenem binding site, thereby eliminating a clash that is predicted to occur in wildtype OXA-51. Similarly, in the OXA-51 variant complex, L129 adopts a different rotamer compared to I129 in wildtype OXA-51. This alternative position moves its side chain away from the hydroxyethyl moiety of doripenem and relieves another potential clash between the enzyme and carbapenem substrates. Molecular dynamics simulations of OXA-51 and OXA-51 I129L demonstrate that compared to isoleucine, a leucine at this position greatly favors a rotamer that accommodates the ligand. These results provide a molecular justification for how this substitution generates enhanced binding affinity for carbapenems, and therefore helps explain the prevalence of this substitution in clinical OXA-51 variants.