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

Abstract β-lactamase inhibitors are central in overcoming emerging antibiotic resistance, and boronate-based β-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 β-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 – 250 ms. Furthermore, the displacement of the crucial anion in the active site of the β-lactamase was verified as an essential part of the binding mechanism of substrates and inhibitors. In total, 22 datasets of β lactamase intermediate complexes with high spatial resolution of 1.40 – 2.04 Å and high temporal resolution range of 50 – 10000 ms were obtained, allowing a detailed analysis of the studied processes. Mechanistic details captured here contribute to the understanding of the recently developed class of boronate-based β lactamase inhibitors.