Unanticipated intermediates during isopenicillin N synthase catalysis identified by time-resolved X-ray free-electron laser studies

Abstract Biosynthesis of penicillin and cephalosporin antibiotics involves the oxidation of the tripeptide δ-( l -α-aminoadipoyl)- l -cysteinyl- d -valine (ACV) to give the conformationally strained bicyclic penicillin ring system, as catalysed by the Fe(II)-dependent oxidase isopenicillin N synthase (IPNS). The structures of the precursors for the sequential β-lactam and thiazolidine ring forming steps in IPNS have remained elusive. Here we studied the reaction of anaerobic IPNS:Fe(II):ACV microcrystals with O 2 using time-resolved serial femtosecond crystallography and X-ray emission spectroscopy. High-resolution structures reveal that ACV cysteinyl oxidation produces a thioaldehyde that unexpectedly does not coordinate the iron centre. The use of deuterium-labelled ACV enabled the observation of a monocyclic β-lactam with restoration of the Fe–S bond. Together with mutagenesis, spectroscopic and computational studies, the results support Fe(IV)-mediated penicillin formation involving active-site water molecules. 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.

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Journal
Nature Catalysis
Published
2026-10-09
DOI
https://doi.org/10.1038/s41929-026-01618-4
Primary Topic
Microbial Natural Products and Biosynthesis
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article
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article

Unanticipated intermediates during isopenicillin N synthase catalysis identified by time-resolved X-ray free-electron laser studies

Sebastian Dehe, Robert Bosman, Leland Bruce Gee, Jordan Welsh et al.
Nature Catalysis
Microbial Natural Products and Biosynthesis
article

Unanticipated intermediates during isopenicillin N synthase catalysis identified by time-resolved X-ray free-electron laser studies

Sebastian Dehe, Robert Bosman, Leland Bruce Gee, Jordan Welsh, Carol V. Robinson, Tiankun Zhou, Patrick Rabe, Aaron S. Brewster, James R. D. Montgomery, Humberto Sánchez, Hiroki Makita, Mark S. Hunter, Jan F Kern, Allen Milster Orville, Medhanjali Dasgupta, Midhun George Thomas, William K. Myers, J. Linyard, Shyam Basak, Margaret Doyle, Kyle D. Sutherlin, M.A. McDonough, Pierre Aller, Jos J. A. G. Kamps, Stephen M. Keable, J. Frederick W. Mosselmans, Isabel Bogacz, Christopher John Schofield, Vittal K. Yachandra, In‐Sik Kim, Coral Mycroft, Abraham Olusegun Oluwole, Christina Redfield, Kensuke Tono, Anastasya Shilova, Pauline A. Lang, Mariska de Munnik, Christo Z. Christov, Vandana Tiwari, Ian J. Clifton, Jürgen Brem, Edgars Sūna, Uwe Bergmann, Frédéric Poitevin, Agata Butryn, Karina Calvopiña, Danny Axford, David Moreau, Nicholas K. Sauter, Rebecca Southwart, Corey J. Kaminsky, Roberto Alonso‐Mori, Philipp S. Simon, Robin Leslie Owen, Junko Yano, Asmit Bhowmick, Daniel W. Paley⧓, Shusaku Hayama, Jaehyun Park, Daniel J. Rosenberg, Alexander T. Stead, Kuntal Chatterjee, Arjun Cheema, Anthony Tumber, Shigeki Owada, Nicholas Devenish, Malkit Sami, Mihails Kazaks, Frederick J. H. Foster
article en

Abstract

Abstract Biosynthesis of penicillin and cephalosporin antibiotics involves the oxidation of the tripeptide δ-( l -α-aminoadipoyl)- l -cysteinyl- d -valine (ACV) to give the conformationally strained bicyclic penicillin ring system, as catalysed by the Fe(II)-dependent oxidase isopenicillin N synthase (IPNS). The structures of the precursors for the sequential β-lactam and thiazolidine ring forming steps in IPNS have remained elusive. Here we studied the reaction of anaerobic IPNS:Fe(II):ACV microcrystals with O 2 using time-resolved serial femtosecond crystallography and X-ray emission spectroscopy. High-resolution structures reveal that ACV cysteinyl oxidation produces a thioaldehyde that unexpectedly does not coordinate the iron centre. The use of deuterium-labelled ACV enabled the observation of a monocyclic β-lactam with restoration of the Fe–S bond. Together with mutagenesis, spectroscopic and computational studies, the results support Fe(IV)-mediated penicillin formation involving active-site water molecules. 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.

Nature Catalysis
Michigan Technological University (US), Pohang University of Science and Technology (KR), University of Wisconsin–Madison (US), Lawrence Berkeley National Laboratory (US), SLAC National Accelerator Laboratory (US), Diamond Light Source (GB), University of Oxford (GB), Research Complex at Harwell (GB), Latvijas Organiskās Sintēzes Institūts (LV), SPring-8 (JP), RIKEN (JP), Japan Synchrotron Radiation Research Institute (JP), Linac Coherent Light Source (US), Pohang Accelerator Laboratory
Openalex Percentile: Top 14%
Microbial Natural Products and Biosynthesis
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