An international team of chemists has filmed the exact moment an enzyme builds the molecular ring that makes penicillin work, closing a mechanistic question that has stood open for more than four decades. Using ultrafast pulses from an X-ray free-electron laser, researchers led by the University of Oxford's Department of Chemistry captured a frame-by-frame "molecular movie" of isopenicillin N synthase (IPNS), the enzyme every penicillin-producing fungus uses to stitch a four-atom beta-lactam ring into its antibiotic scaffold. The work was published this week in Nature Catalysis.
The team deposited anaerobic microcrystals of the enzyme, bound to its natural peptide substrate, onto a moving tape and let oxygen flood in to trigger the reaction, collecting thousands of ultrafast X-ray snapshots as it unfolded. Stitched together, those frames revealed two fleeting structures no one had directly observed before: a short-lived thioaldehyde intermediate and, just after it, the first monocyclic beta-lactam ring on the path to penicillin's finished scaffold. Water molecules positioned inside the enzyme's active site, along with small shifts in the protein's own shape, help steer the chemistry at each step.
A question open since the 1980s
Scientists have known the broad outline of IPNS chemistry since the 1980s, but the identity and order of its fastest intermediates — each surviving for a tiny fraction of a second — had eluded conventional crystallography. The new study builds on a 2021 time-resolved X-ray study by an overlapping Oxford-led team that first showed oxygen binding inside the enzyme; this time, the collaboration — which also drew on beamlines at the UK's Diamond Light Source, South Korea's PAL-XFEL, and the SLAC and Lawrence Berkeley national laboratories in the United States — pushed further into the reaction's core steps.
The stakes extend beyond historical curiosity. Beta-lactams, the drug class that includes penicillins and cephalosporins, remain among the most widely prescribed antibiotics on Earth, even as resistance erodes their effectiveness. "Penicillin has shaped modern medicine, but there is still much to learn" about how nature actually assembles it, said Christopher Schofield, an Oxford chemistry professor and the study's senior author. First author Patrick Rabe said the atomic-level detail could eventually feed back into drug design: "By understanding this process in atomic detail, we can begin to think about engineering these enzymes."
"It is a great example of how time-resolved structural biology can uncover new principles of enzyme function."
Dr. Allen M. Orville, Diamond Light Source XFEL Hub, study co-author
It's worth being clear what this study does and doesn't show. It is a structural, mechanistic paper — a peer-reviewed description of how one enzyme behaves in a lab reaction, not a new antibiotic, a drug trial, or evidence that resistant infections can now be treated differently. No patients, animals, or whole cells were involved; the experiments used purified enzyme and substrate in vitro. Translating this clearer picture of IPNS chemistry into new antibiotics — something the authors hope to pursue by engineering the enzyme — would require years of further synthetic and biological work.
The team says the same time-resolved XFEL approach could now be turned on other enzymes that build complex natural products through similarly short-lived intermediates, potentially opening a broader window onto how nature's chemistry works at timescales too fast for older imaging methods to catch.
