Direct Observation of the Mechanism of Antibiotic Resistance by Mix-and-Inject at the European XFEL

Abstract

In this study, we follow the diffusion and buildup of occupancy of the substrate ceftriaxone in M. tuberculosis β-lactamase BlaC microcrystals by structural analysis of the enzyme substrate complex at single millisecond time resolution. We also show the binding and the reaction of an inhibitor, sulbactam, on a slower millisecond time scale. We use the ‘mix-and-inject’ technique to initiate these reactions by diffusion, and determine the resulting structures by serial crystallography using ultrafast, intense X-ray pulses from the European XFEL (EuXFEL) arriving at MHz repetition rates. Here, we show how to use the EuXFEL pulse structure to dramatically increase the size of the data set and thereby the quality and time resolution of “molecular movies” which unravel ligand binding and enzymatically catalyzed reactions. This shows the great potential for the EuXFEL as a tool for biomedically relevant research, particularly, as shown here, for investigating bacterial antibiotic resistance.One Sentence SummaryDirect observation of fast ligand binding in a biomedically relevant enzyme at near atomic resolution with MHz X-ray pulses at the European XFEL.

Mapping Enzyme Landscapes by Time-Resolved Crystallography with Synchrotron and X-Ray Free Electron Laser Light

“…A new generation of time-resolved experiments at synchrotrons and XFELs has expanded that information into the nonequilibrium domain, including enzyme catalysis. As the ability to rapidly collect X-ray diffraction data sets at many time points improves (86), the resulting time series of electron density maps will provide unprecedentedly detailed views of enzymes sampling nonequilibrium ensembles during catalysis. The future of time-resolved structural enzymology is closely tied to the rapidly evolving technical capabilities of synchrotron and XFEL light sources, as well as the continued development of analytical methods capable of parsing the complicated, timedependent electron density maps that can now be generated.…”

“…One advantage of MISC is that the technique is applicable to many enzymes whose substrates are small enough to soak into a crystal, which include a large number of candidate systems that cannot be phototriggered. MISC has been successfully used to observe catalysis in β-lactamase (83,86), isocyanide hydratase (ICH) (25), and cytochrome c oxidase (50). Additional enzyme MISC experiments are in progress at the time of this writing.…”