Ingenious ‘molecular movie’ finally reveals how penicillin is made
More than 80 years after scientists at the University of Oxford turned penicillin into a lifesaving medicine, researchers have revealed previously unseen stages in how nature builds its antibiotic structure. The findings have been published today in Nature Catalysis.
Using X-ray free electron laser experiments, the researchers captured structural snapshots of key intermediates during the reaction that produces penicillin. Image credit: University of Oxford/Greg Stewart/SLAC National Accelerator Laboratory.
Ever since penicillin was developed into a working drug at the University of Oxford in the early 1940’s, β-lactam antibiotics have been among the most important medicines for treating bacterial infection. Their activity depends on a special ring - the β-lactam ring - a highly strained chemical ring system that interferes with bacterial cell wall synthesis, ultimately causing the cell wall to fail and the bacteria to die.
Scientists have been studying how nature constructs this β-lactam ring for decades, but the key fleeting reaction intermediates have been too difficult to observe directly. Researchers from the University of Oxford and international collaborators have revealed previously unseen rapid chemical stages in the formation of β-lactam antibiotics like penicillin, offering insights that can support future antibiotic development.
“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.”
Rising rates of antimicrobial resistance- a process in which bacteria, fungi and other microorganisms evolve to survive the medicines designed to kill them, are undermining the effectiveness of existing antibiotics. With too few new antibiotics in development, understanding how nature builds these molecules is an important step towards replenishing the antibiotic pipeline.
In the new study, researchers from the University of Oxford in collaboration with partners from Diamond Light Source, Lawrence Berkeley National Laboratory, PAL-XFEL and SLAC National Accelerator Laboratory have used X-ray free-electron lasers to observe the enzyme isopenicillin N synthase (IPNS) as it converts its linear peptide substrate into the ring system of penicillin.
The new study shows, in unprecedented detail, how the enzyme IPNS achieves an exceptionally complex transformation in a single step. This resolves a long-standing mechanistic question that has remained unanswered for more than four decades.
Rather than relying on static X-ray crystallographic structures of the enzyme, the researchers followed the reaction in real time using ultrafast X-ray free-electron laser (XFEL) experiments.
The team captured several rapid stages in the reaction. These included a thioaldehyde intermediate formed just before the β-lactam ring is created, and a monocyclic β-lactam intermediate, representing the first ring-shaped structure on the way to forming the complete penicillin scaffold. These steps provide the clearest picture yet of how the IPNS enzyme assembles the penicillin scaffold.
The work also showed that water molecules inside the enzyme play a key role in guiding the reaction. Subtle movements throughout the enzyme help guide these chemical steps, showing that both the enzyme's shape and its chemistry work together to control penicillin formation.
To capture the reaction in real time, the researchers used a system in which thousands of tiny droplets containing anaerobic enzyme microcrystals were deposited onto a moving 2 mm wide tape. As the tape entered an oxygen filled chamber, oxygen rapidly diffused into the crystals and initiated the reaction simultaneously across the sample. By precisely controlling the speed of the tape, the researchers could determine 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 allowed the team to build a frame-by-frame ‘molecular movie’ of penicillin biosynthesis. This method allows individual reaction intermediates that exist only for tiny fractions of a second to be observed at atomic resolution, and under physiological temperature and pressure, before they disappear.
Oxford has played a central role in the history of penicillin since the pioneering work of Nobel prize-winning scientists Prof Howard Florey and Sir Ernst Chain, and colleagues, who developed penicillin into the first clinically useful antibiotic during the Second World War. This new study ultimately builds on the work of Nobel prize-winning Oxford chemist Dorothy Hodgkin, who first solved the structure of penicillin in 1945 using X-ray crystallography.
“The ability to capture structural snapshots over milliseconds to seconds of reaction time allows us to connect enzyme motion, iron chemistry and water-mediated proton transfer. This gives us a much richer view of how IPNS controls a difficult chemical transformation. By understanding this process in atomic detail, we can begin to think about engineering these enzymes to produce new or improved antibiotic scaffolds.”
Beyond penicillin, IPNS belongs to a large family of iron-dependent oxygenase enzymes involved in human biology, including ones that enable us to sense and respond to changes in oxygen availability. The mechanistic insights from this work therefore have implications far beyond antibiotics, offering new principles for enzyme engineering and catalyst design.
Today, one in six bacterial infections is resistant to antibiotics. Rising resistance threatens decades of progress in cancer care, transplant surgery and other areas of modern medicine, while also placing pressure on economies, health systems and food production.
The paper ‘Unanticipated intermediates during isopenicillin N synthase catalysis identified by time-resolved X-ray free-electron laser studies' has been published in Nature Catalysis.
The work was led by researchers in the Department of Chemistry, University of Oxford, in collaboration with Lawrence Berkeley National Laboratory, SLAC National Accelerator Laboratory and international partners. Experiments were carried out using X-ray free-electron lasers at LCLS (USA), PAL-XFEL (Republic of Korea), SACLA (Japan) and Diamond Light Source (UK).
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