Researchers used SLAC\u2019s Linac Coherent Light Source to study the AspH enzyme \u2013 a potential therapeutic target for cancer treatment \u2013 and the isopenicillin N synthase (IPNS) enzyme, which is essential for creating all of the medicinally important penicillin antibiotics.By leveraging the power of ultrabright, ultrashort X-ray bursts, they found that these enzymes catalyze reactions in surprising ways.If scientists can better understand the structure and function of these enzymes, they could potentially use them as biomarkers and therapeutic targets for cancer, or for a method to develop new antibiotics.<\/p>\n
Newswise \u2014 Within biology, one group of enzymes, called 2-oxoglutarate-dependent oxygenases, is responsible for catalyzing a very wide range of reactions that play a role in everything from how genetic code is used by cells to synthesizing antibiotics.<\/p>\n
Within the past several years, researchers have used ultrashort, powerful light pulses from X-ray free-electron lasers, including the Linac Coherent Light Source (LCLS) at the Department of Energy\u2019s\u00a0SLAC National Accelerator Laboratory<\/a>, to better understand just how these enzymes catalyze reactions.<\/p>\n In two recent papers, researchers used LCLS to study the AspH enzyme\u00a0\u2013\u00a0a potential therapeutic target for cancer treatment\u00a0\u2013\u00a0and the isopenicillin N synthase (IPNS) enzyme, which is essential for creating penicillin. By examining those reactions step-by-step at mere quadrillionths of a second, they found that these enzymes activate reactions in surprising ways\u00a0\u2013 leading to new insights that could inspire novel therapeutics and drug development.\u00a0<\/p>\n \u201cWe have gone from examining static structures to making \u2018molecular movies\u2019 of the dynamics of biological systems,\u201d said\u00a0Robert Alonso-Mori<\/a>, senior scientist at SLAC. \u201cIt has really given us an understanding of the function of proteins.\u201d<\/p>\n The research was led by\u00a0Patrick Rabe<\/a> and\u00a0Christopher Schofield<\/a>,\u00a0research group leaders at the University of Oxford, UK, in close collaboration with Jan Kern<\/a>, senior scientist at DOE\u2019s Lawrence Berkeley National Laboratory (Berkeley Lab). The research was published in\u00a0Nature Communications<\/a>\u00a0and Nature Catalysis<\/a>.<\/p>\n Using X-ray free-electron lasers to identify potential pathways for therapeutics<\/p>\n In humans, the AspH enzyme is encoded by the AspH gene. Scientists have found that levels of AspH are increased in multiple cancers, especially the hard-to-treat brain cancer called glioblastoma. By examining the structure and function of AspH and how it is regulated, scientists could potentially use the enzyme as both a biomarker and a therapeutic target for cancer treatment.<\/p>\n Penicillin has a remarkable structure made up of two reactive rings, but as a consequence, it cannot be made by chemical synthesis. Instead, it is made by fermentation in microbial cells. During the process of manufacturing penicillin in cells, the IPNS enzyme catalyzes a vital early-stage reaction. Analyzing just how this reaction works could help scientists find a way to create synthetic modified penicillin that could overcome antibiotic resistance to better treat bacterial infections.<\/p>\n Researchers have studied how these enzymes catalyze reactions for years, but capturing intermediates \u2013 the molecules produced and then consumed in the middle of a reaction \u2013 has been challenging.<\/p>\n At LCLS, researchers use macromolecular crystallography with femtosecond pulses to observe what exactly happens during these reactions. Leveraging extremely bright and short X-ray bursts, they can do so in atomic detail, on timescales of millionths of a billionth of a second, and at room temperature without damaging the biological samples.\u00a0<\/p>\n At the\u00a0Macromolecular Femtosecond Crystallography<\/a> (MFX) instrument, researchers create microcrystal samples of the enzymes and run them through a system that injects oxygen to induce the reaction. They then examine how X-rays from LCLS scatter off the sample in real time, turning those data into molecular movies of the reactions. “We have been working for many years to develop experimental protocols that allow us to precisely start the reaction in these fascinating enzymes and watch them perform complicated chemistry step by step in real time,” Kern said.<\/p>\n \u201cWe wanted to understand specifically how enzymes are controlling the intermediates,\u201d Rabe said.<\/p>\n \u2018Molecular movies\u2019 reveal surprising results<\/p>\n For both enzymes, researchers knew that iron played a key role in the reactions \u2013 but were surprised by how the steps played out. \u201cOur observations from these experiments were not predicted by sophisticated calculations,\u201d Schofield said.<\/p>\n For AspH, the researchers saw how the enzyme binds to a substrate and activates oxygen during catalysis. In particular, they saw that an iron-bound water molecule remains stable throughout the reaction, a factor that appeared to be important for catalysis. The research team knew that iron-bound water was essential for catalysis, but seeing this stability \u2013 and showing that if it\u2019s disrupted, the reaction won\u2019t proceed \u2013 could help scientists develop a pathway to treating AspH activity in cancer cells.\u00a0<\/p>\n They also found that AspH only uses two metal-coordinating residues \u2013 amino acids that bind to specific sites in proteins \u2013 instead of three residues usually seen in related proteins. Scientists could ultimately use this information to engineer new proteins that could modify peptides for therapeutic benefit.<\/p>\n In the case of IPNS, the researchers took snapshots of the intermediates during the synthesis of the penicillin rings. They found that large parts of the enzyme are involved in this reaction \u2013 not just the iron-containing site with the substrate \u2013 and that there are key water molecules involved. In fact, the water molecules are used to control the outcome of the reaction.\u00a0<\/p>\n \u201cThat was very surprising to us,\u201d Schofield said. \u201cBoth that regions away from the active site are important and that these active site water molecules were crucial to catalysis.\u201d This step-by-step framework of IPNS catalysis could help scientists engineer enzymes to make penicillin more efficiently or target disease in new ways.\u00a0<\/p>\n A crucial next step is to understand the intricate involvement of water in catalysis. The team also wants to create compounds that either promote or inhibit the activity of these enzymes \u2013 as a potential cancer treatment, for example \u2013 to understand them even more.<\/p>\n International, multidisciplinary collaboration drives innovation<\/p>\n The team has worked together for years on these projects, with Rabe and Schofield traveling from the United Kingdom to work with\u00a0Alonso-Mori at SLAC and Kern at Berkeley Lab. The team also includes researchers across disciplines who provide the wide range of expertise needed to develop and use the technology necessary to understand nature at such a small scale and work toward new therapies for hard-to-treat diseases.<\/p>\n \u201cIt\u2019s a really nice consortium of biologists, chemists, physicists, computational analysts and engineers that all come together to study this, because we all need each other,\u201d Rabe said.\u00a0<\/p>\n \u201cMany of us dreamt about having experiments like this decades ago, so, to have these results and to be able to move forward with this research is a dream come true,\u201d Schofield said.\u00a0<\/p>\n The research was in part funded by DOE\u2019s Office of Science and the National Institutes of Health. In addition to researchers from SLAC, Berkeley Lab, and the University of Oxford, the research collaborations included scientists from the U.S., U.K., Republic of Korea, Japan and Latvia. X-ray data were collected at LCLS, a DOE Office of Science user facility; PAL-XFEL, Republic of Korea; SACLA, Japan; and Diamond Light Source, U.K. \u00a0<\/p>\n Citation: De Munnik et al., Nature Communications, 25 February 2026 (10.1038\/s41467-026-69425-w<\/a>);\u00a0Rabe et al., Nature Catalysis, 09 October 2026 (10.1038\/s41929-026-01618-4<\/a>.<\/p>\n About SLAC<\/p>\n SLAC National Accelerator Laboratory explores how the universe works at the biggest,\u00a0smallest\u00a0and fastest scales and invents powerful tools used by researchers around the globe. As world leaders in ultrafast science and bold explorers of the physics of the universe, we forge new ground in understanding our origins and building a healthier and more sustainable future. Our\u00a0discovery and innovation<\/a> help develop new materials and chemical processes and open unprecedented views of the cosmos and life\u2019s most delicate machinery. Building on more than 60 years of visionary research, we help shape the future by advancing areas such as quantum technology, scientific computing and the development of next-generation accelerators.<\/p>\n