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

Abstract

The threat posed by the chromosomally encoded class D β-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 β-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 β-lactams against this troublesome pathogen.

FIG 1

OXA-51 aligned to OXA-24/40/doripenem. The overall main-chain structure of OXA-51 (blue) matches very closely that of OXA-24/40 (cyan). The structure of doripenem (cyan, center top) indicates the position of the active site. Both structures contain three loops surrounding the active site: the P loop and the β5β6 loop (β8β9 in OXA-51) contribute large hydrophobic residues that cap the top of the active site, and the omega loop forms the front wall.

FIG 2

Alignment of OXA-66 and eight clinical variants. OXA-66 and several variants containing one, two, or three substitutions were aligned using ClustalW. The red bar between residues 25 and 26 represents the site of proteolytic digestion for removal of the export sequence. Key active-site residues are highlighted with blue, and the substitutions are highlighted with green. OXA-PQ/WL is a variant without an OXA number and is the P130Q/W222L double mutant of OXA-66 (NCBI RefSeq number WP_001022759).

FIG 3

Models of OXA-172 and OXA-173. To help explain the potential structural effects of the substitutions that resulted in enhancement of hydrolytic activities, we constructed models of variants that contained those substitutions. (A) Model of OXA-66 I129V/W222L (OXA-172; magenta) superposed with a model of OXA-66 wild type (blue) and OXA-24/40/doripenem (PDB code 3PAE) (cyan; ligand only). (B) Model of OXA-66 I129V/W222L/P226L (OXA-173; yellow) superposed with a model of OXA-66 wild type (blue) and OXA-225/ceftazidime (PDB code 4X55) (salmon; ligand only). dori, doripenem; ceftaz, ceftazidime.

FIG 4

Rotamer position of I129 in OXA-66 and OXA-109. The χ₁ torsion angle for I129 was plotted as a function of time for OXA-66 (blue) and OXA-109 (green). Rotamers (approximate structures shown at right) were binned as follows: gauche (−), −120° < χ₁ <0°; gauche (+), 0° < χ₁ <120°; trans, −180° < χ₁ < −120° and 120° < χ₁ <180°. For purposes of clarity, χ₁ values between −120° and −180° were shifted to the top of the graph by adding 360°.

FIG 5

Comparison of the distance between the side chain of W222 and the expected position of doripenem in OXA-66 and OXA-109. The distance between the C-1 methyl carbon of the pyrroline ring of doripenem and the Cη₂ of W222 was measured for each frame of the OXA-66 trajectory (blue) and the OXA-109 trajectory (green). To estimate the position of doripenem in the active sites of OXA-66 and OXA-109, the structures were aligned with the structure of OXA-24/40/doripenem (PDB code 3PAE). Representative snapshots of the position of W222 pointing away from the binding site (OXA-109; green) or toward the binding site (OXA-66; blue) are shown at right.

FIG 6

Stereo view of the superposition of frames from the molecular dynamics simulation of OXA-66 (blue) and OXA-109 (green) with acyl-doripenem from the crystal structure of OXA-24/40 (PDB 3PAE) (cyan; OXA-24/40 protein not shown). In OXA-66, I129 and W222 predominantly occupy positions that would be expected to clash extensively with the hydroxyethyl (I129) and pyrroline C-1 methyl (W222) groups of doripenem. The introduction of the P130Q substitution in OXA-109 leads to alternative conformations of both residues that greatly alleviate the steric clash.