14 September 2021
Photo: European XFEL
The detector AGIPD at the European XFEL used for the experiment.
Using the X-ray laser European XFEL an international team of researchers, including scientists from DESY and Universität Hamburg, has successfully filmed a reaction step that is important for the development of antibiotic resistance. The molecular film captures the very rapid reaction of the enzyme beta-lactamase from tuberculosis bacteria with the cephalosporin antibiotic ceftriaxone in slow motion. The team reports the results in IUCrJ, the journal of the International Union of Crystallography.
Antibiotic resistance occurs, in part, when bacteria acquire the ability to inactivate the antibiotics used against them. Many resistant bacteria produce the enzyme beta-lactamase. This enzyme can render the most commonly used beta-lactam antibiotics ineffective. The team, led by Prof. Marius Schmidt of the University of Wisconsin-Milwaukee, have now studied the first step of this inactivation at the European XFEL, observing how the antibiotic binds to the beta-lactamase from resistant tuberculosis bacteria within milliseconds (thousandths of a second).
The researchers also investigated how the enzyme inhibitor sulbactam reacts with the bacterial enzyme. Sulbactam, administered at the same time as the antibiotics, binds to the bacterial beta-lactamase, thereby blocking it. As a result the enzyme is unable to inactivate the antibiotics whose effectiveness is restored this way.
XFELs enable use of small crystals
Up to now, crystals are a requirement to study biomolecules using X-rays. Such crystals are made up of millions of individual enzyme molecules that are formed into a regular arrangement in three dimensions. If the crystals are big enough then bright X-ray diffraction patterns can be measured, which are used to reveal the spatial structure of the individual molecules and compounds in the crystal.
“We wish to make the X-ray measurements at specific times after the enzyme molecules in the crystals are brought in contact with the antibiotic,” explains co-author Prof. Henry Chapman from DESY and Universität Hamburg, who worls at the Center for Free-Electron Laser Science CFEL. “However, when crystals are big enough for good X-ray measurements it can take several seconds for the antibiotic solution to diffuse through the crystal, smearing out the view of molecular changes that occur at faster timescales.” It was only with the advent of the X-ray free-electron lasers or XFELs, using intense pulsed beams that enable exposures that are thousands of times greater than could be tolerated before, that small enough crystals could be used. “Then, diffusion is quicker than the actual reaction so that details of the reaction can be observed,” says Chapman, who is also a member of the Cluster of Excellence "CUI: Advanced Imaging of Matter".
Important tool for biological research
The special mix-and-inject method used in the study requires custom-made injectors, which were provided by the group of Prof. Lois Pollack from Cornell University for use at the European XFEL. The method enables atomically accurate slow-motion imaging of fast biological processes in enzymes and other biomolecules.
“Enzymes such as beta-lactamase are of great importance for medicine,” explains Schmidt. “X-ray lasers like the European XFEL now make it possible to study much smaller crystals than in the past. Our experiment, together with previous results, has shown how X-ray lasers can be used as an important tool for biological research in the future,” he adds.
Scientists from the University of Wisconsin-Milwaukee, Cornell University, Arizona State University, Lawrence Livermore National Laboratory, Rice University, University of California San Francisco, La Trobe University, Spanish National Research Council, University of Hamburg, European XFEL and DESY contributed to this work. Text: DESY, ed. / Press release XFEL and Video
Suraj Pandey, Marius Schmidt et al.
"Observation of Substrate Diffusion and Ligand Binding in Enzyme Crystals using High Repetition Rate Mix-and-Inject Serial Crystallography"