Researchers at the University of Oxford, working with Diamond Light Source, Lawrence Berkeley National Laboratory, PAL-XFEL and SLAC National Accelerator Laboratory, have captured previously unobserved chemical intermediates in penicillin biosynthesis — including a thioaldehyde intermediate and a monocyclic β-lactam — using X-ray free-electron laser (XFEL) crystallography. The work resolves a mechanistic question that has stood for more than four decades and appears in Nature Catalysis under the title "Unanticipated intermediates during isopenicillin N synthase catalysis identified by time-resolved X-ray free-electron laser studies."

The team studied isopenicillin N synthase (IPNS), the iron-dependent oxygenase that converts a linear peptide substrate into isopenicillin N (IPN) — the fused four-membered β-lactam and five-membered thiazolidine ring system characteristic of penicillins. Instead of relying on static X-ray crystallographic structures, the researchers tracked the reaction in real time at atomic resolution, under physiological temperature and pressure.

How did they capture millisecond intermediates?

The experimental design is the enabling step. Thousands of tiny droplets containing anaerobic enzyme microcrystals were deposited onto a moving tape 2 mm wide. When the tape entered an oxygen-filled chamber, oxygen diffused rapidly into the crystals and started the reaction simultaneously across the sample. By controlling tape speed, the researchers set how long each crystal reacted before reaching the X-ray interaction point, where an ultrafast XFEL pulse recorded an atomic-resolution snapshot.

Combining thousands of these snapshots produced a frame-by-frame "molecular movie" of penicillin biosynthesis, capturing intermediates that exist for fractions of a second. Two intermediates stand out:

• A thioaldehyde intermediate, formed just before the β-lactam ring is created
• A monocyclic β-lactam intermediate, the first ring-shaped structure on the path to the complete penicillin scaffold

The results show how IPNS achieves an exceptionally complex transformation in a single step, with subtle enzyme movements — and active-site water molecules — guiding the chemistry. The authors write that their studies "reveal unexpected insight into the IPNS reaction mechanism including the vital roles of active-site water molecules." Water-mediated proton transfer, iron chemistry and enzyme motion together control the transformation.

Why does this matter for antibiotic R&D?

Rising antimicrobial resistance is eroding the β-lactam arsenal. Citing WHO figures, the authors note that one in six bacterial infections is now resistant to antibiotics, while too few new antibiotics sit in the development pipeline. β-lactams remain among the most important medicines for treating bacterial infections; their activity depends on the strained β-lactam ring, which disrupts bacterial cell wall synthesis.

Christopher Schofield, PhD, Professor of Chemistry at Oxford and co-senior author, said: "Penicillin has shaped modern medicine, but there is still much to learn about how nature builds this important antibiotic structure. By capturing these fleeting steps, we can better understand how enzymes control complex chemistry with remarkable precision … As rates of antimicrobial resistance continue to rise, understanding this process will ultimately help us make existing antibiotics more efficient and design new antibiotic structures."

First author Patrick Rabe, PhD, a Wellcome Career Development Award Investigator at Oxford, framed the engineering implication directly: "By understanding this process in atomic detail, we can begin to think about engineering these enzymes to produce new or improved antibiotic scaffolds." He noted that structural snapshots spanning milliseconds to seconds connect enzyme motion, iron chemistry and water-mediated proton transfer.

Beyond penicillin?

The significance extends past antibacterials. IPNS belongs to a large family of iron-dependent oxygenases involved in human biology, including enzymes that enable cells to sense and respond to changes in oxygen availability. The team stated in the paper: "These findings provide structural insight into IPNS catalysis, inform on the wider Fe(II) oxygenase superfamily and highlight the power of X-ray free-electron laser methods for resolving transient enzymatic intermediates."

Allen M Orville, PhD, group leader of the XFEL Hub at Diamond Light Source, said the collaboration "helped bring together the experimental approaches needed to capture these fleeting stages of enzyme catalysis" and called the work "a great example of how time-resolved structural biology can uncover new principles of enzyme function and, ultimately, inform the design of new catalysts and therapeutics."

What is the historical through-line?

The study builds directly on the legacy of Nobel laureate Dorothy Hodgkin, PhD, who solved the structure of penicillin at Oxford in 1945 using X-ray crystallography — and on the university's role in developing penicillin into a working drug in the early 1940s. Eighty years later, XFEL instrumentation has moved the field from static structures to real-time observation of catalysis.

For R&D managers, the work signals that time-resolved XFEL methods — now accessible through hubs at user facilities such as Diamond — can interrogate fleeting intermediates in enzyme mechanisms that conventional crystallography cannot reach, with applications in biosynthesis, enzyme engineering and catalyst design well beyond antibiotics.