Arginine Modulates Carbapenem Deactivation by OXA-24/40 in Acinetobacter baumannii.

The resistance of Gram-negative bacteria to ß-lactam antibiotics stems mainly from ß-lactamase proteins that hydrolytically deactivate the ß-lactams. Of particular concern are the ß-lactamases that can deactivate a class of ß-lactams known as carbapenems. Carbapenems are among the few anti-infectives that can treat multi-drug resistant bacterial infections. Revealing the mechanisms of their deactivation by ß-lactamases is a necessary step for preserving their therapeutic value. Here, we present NMR investigations of OXA-24/40, a carbapenem-hydrolyzing Class D ß-lactamase (CHDL) expressed in the gram-negative pathogen, Acinetobacter baumannii. Using rapid data acquisition methods, we were able to study the "real-time" deactivation of the carbapenem known as doripenem by OXA-24/40. Our results indicate that OXA-24/40 has two deactivation mechanisms: canonical hydrolytic cleavage, and a distinct mechanism that produces a ß-lactone product that has weak affinity for the OXA-24/40 active site. The mechanisms issue from distinct active site environments poised either for hydrolysis or ß-lactone formation. Mutagenesis reveals that R261, a conserved active site arginine, stabilizes the active site environment enabling ß-lactone formation. Our results have implications not only for OXA-24/40, but the larger family of CHDLs now challenging clinical settings on a global scale.

NMR Relaxation Dispersion Reveals Macrocycle Breathing Dynamics in a Cyclodextrin-based Rotaxane.

A distinctive feature of mechanically interlocked molecules (MIMs) is the relative motion between the mechanically bonded components, and often it is the functional basis for artificial molecular machines and new functional materials. Optimization of machine or materials performance requires knowledge of the underlying atomic-level mechanisms that control the motion. The field of biomolecular NMR spectroscopy has developed a diverse set of pulse schemes that can characterize molecular dynamics over a broad time scale, but these techniques have not yet been used to characterize the motion within MIMs. This study reports the first observation of NMR relaxation dispersion related to MIM motion. The rotary (pirouette) motion of α-cyclodextrin (αCD) wheels was characterized in a complementary pair of rotaxanes with pirouetting switched ON or OFF. 13C and 1H NMR relaxation dispersion measurements reveal previously unknown exchange dynamics for the αCD wheels in the pirouette-ON rotaxane with a rate constant of 2200 s-1 at 298 K and an activation barrier of ΔF‡ = 43 ± 3 kJ/mol. The exchange dynamics disappear in the pirouette-OFF rotaxane, demonstrating their switchable nature. The 13C and 1H sites exhibiting relaxation dispersion suggest that the exchange involves "macrocycle breathing", in which the αCD wheel fluctuates between a contracted or expanded state, the latter enabling diffusive rotary motion about the axle. The substantial insight from these NMR relaxation dispersion methods suggests similar dynamic NMR methods can illuminate the fast time scale (microsecond to millisecond) mechanisms of intercomponent motion in a wide range of MIMs.

High affinity threading of a new tetralactam macrocycle in water by fluorescent deep-red and near-infrared squaraine dyes.

A new tetralactam macrocycle was prepared and found to encapsulate deep-red and near-infrared squaraine and croconaine dyes in water with tunable threading kinetics. The new supramolecular paradigm of guest back-folding was used to increase macrocycle/squaraine affinity by 370-fold and achieve an association constant of 2.8 × 109 M-1.