In recent years, researchers have used ultrabrief, powerful light pulses from a X-ray free-electron laser (XFEL), including the Linac Coherent Light Source (LCLS) at the Department of Energy’s SLAC National Accelerator Laboratory, to better understand how biologically important enzymes catalyze reactions. Led by Patrick Rabe and Christopher Schofield at the University of Oxford, in close collaboration with Molecular Biophysics and Integrated Bioimaging (MBIB) Division senior scientist Jan Kern, the team examined the the AspH enzyme, a potential therapeutic target for cancer treatment, and the IPNS enzyme, which is essential for creating penicillin. By examining those reactions step-by-step at mere quadrillionths of a second, the team found that these enzymes activate reactions in surprising ways, leading to new insights that could inspire novel therapeutics and drug development.
The pair of studies, published in Nature Communications and Nature Catalysis, respectively, each combined two cutting-edge techniques in a single experiment. The first, time-resolved serial femtosecond crystallography (tr-SFX), uses an XFEL to capture atomic-resolution “movie frames” of an enzyme as it works at room temperature. Rather than freezing a single static structure, the researchers reacted tiny anaerobic crystals with oxygen and captured multiple time points, assembling a sequence of the reaction. The second technique, X-ray emission spectroscopy (XES), was run in parallel to track the iron atom’s oxidation state at each time point.
This integrated tr-SFX/XES approach offers a template for studying transient intermediates in many other enzymes, especially metal-containing ones. No other current method provides comparable high-resolution structural detail of such short-lived catalytic states, making it broadly valuable across structural biology.
In addition to Kern, fellow MBIB researchers including Asmit Bhowmick, Aaron Brewster, Margaret Doyle, David Mittan-Moreau, Daniel Paley, Nicholas Sauter, Philipp Simon, Vittal Yachandra, and Junko Yano contributed the capabilities for room temperature X-ray diffraction and X-ray emission spectroscopy collection, the analysis of the spectroscopy data and tools for processing of the X-ray diffraction data, and computational infrastructure, drawing on national user facilities. Essential for a successful experiment was the direct streaming of experimental data to the National Energy Research Scientific Computing Center (NERSC) facility at Berkeley Lab to allow fast processing and feedback.
