Time-resolved crystallography of boric acid binding to the active site serine of the ß-lactamase CTX-M-14 and subsequent 1,2-diol esterification.

ABSTRACT

The emergence and spread of antibiotic resistance represent a growing threat to public health. Of particular concern is the appearance of ß-lactamases, which are capable to hydrolyze and inactivate the most important class of antibiotics, the ß-lactams. Effective ß-lactamase inhibitors and mechanistic insights into their action are central in overcoming this type of resistance, and in this context boronate-based ß-lactamase inhibitors were just recently approved to treat multidrug-resistant bacteria. Using boric acid as a simplified inhibitor model, time-resolved serial crystallography was employed to obtain mechanistic insights into binding to the active site serine of ß-lactamase CTX-M-14, identifying a reaction time frame of 80-100 ms. In a next step, the subsequent 1,2-diol boric ester formation with glycerol in the active site was monitored proceeding in a time frame of 100-150 ms. Furthermore, the displacement of the crucial anion in the active site of the ß-lactamase was verified as an essential part of the binding mechanism of substrates and inhibitors. In total, 22 datasets of ß-lactamase intermediate complexes with high spatial resolution of 1.40-2.04 Å and high temporal resolution range of 50-10,000 ms were obtained, allowing a detailed analysis of the studied processes. Mechanistic details captured here contribute to the understanding of molecular processes and their time frames in enzymatic reactions. Moreover, we could demonstrate that time-resolved crystallography can serve as an additional tool for identifying and investigating enzymatic reactions.