Clyde Smith

SLAC National Accelerator Laboratory

The last decade has seen the evolution of Acinetobacter baumannii from a microorganism of low clinical importance into a major nosocomial pathogen.

Carbapenem antibiotics have long been drugs of choice for the treatment of A. baumannii infections, however the emergence and spread of carbapenem-resistant isolates has greatly reduced their efficacy. The major mechanism of resistance to carbapenems in A. baumannii is the production of β-lactamases, and the class D OXA-type β-lactamases of A. baumannii have become enzymes of clinical importance. The OXA-51-like enzymes constitute the largest family of class D β-lactamases, yet they remain poorly studied and their importance in conferring carbapenem resistance is not well understood.

We have undertaken the detailed microbiological and kinetic characterization of the chromosomally-encoded A. baumannii OXA-51 β-lactamase. The apo-OXA-51 enzyme was purified to homogeneity and crystallized, and we have determined the three-dimensional structure of this enzyme to 2.0 Å resolution, using data collected at SSRL Beamline12-2.

Kinetic studies show that OXA-51 has low catalytic efficiency for carbapenems, due to a low affinity of the enzyme for these substrates.

Structural studies show that the low affinity results from obstruction of the active site by the side chain of a tryptophan residue conserved in OXA-51-like enzymes. Trp222 presents a transient steric barrier to an incoming substrate, and molecular docking of carbapenems into OXA-51 shows that the Trp222 side chain is able to adopt an alternate conformation such that the substrate can bind in a productive manner. A Trp222Met mutation relieves this steric hindrance and elevates the affinity of the mutant enzyme for carbapenems by 10-fold, significantly increasing the levels of resistance to these antibiotics.

The ability of OXA-51 to evolve into a robust carbapenemase as the result of a single amino acid substitution may, in the near future, elevate the ubiquitous enzymes of the OXA-51 family to the status of the most deleterious A. baumannii carbapenemases, with dire clinical consequences.