Measuring the Dynamics and Kinetics of OXA-58, a Class D Abeta-Lactamase from Acinetobacter Baumannii

OXA-58 is a β-lactamase enzyme that bacteria Acinetobacter baumannii employ as their chief defense mechanism against β-lactam antibiotic treatment. The catalytic activity of OXA-58 and its mutants was monitored as they turned over a series of β-lactam antibiotics. Isothermal titration calorimetry (ITC) showed that the mutation of phenylalanine114 to isoleucine (F114I) had the most detrimental impact on the catalytic efficiency of the enzyme. Isoleucine has a more branched side chain than phenylalanine, which lead to decreased substrate binding and a low catalytic efficiency of F114I mutant. A custom made microfluidic chip coupled to Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) technique was used to study the dynamics of free enzyme and during its reactions with good substrate oxacillin and poor substrate imipenem. The conformational space that OXA-58 explores was constrained in the presence of substrate, suggestive of an induced fit model for catalysis-linked dynamics.