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

Authors

• Patrick Rabe, University of Oxford
• Hiroki Makita, Lawrence Berkeley National Laboratory
• Rebecca Southwart, University of Oxford
• Midhun George Thomas, Michigan Technological University
• Mihails Kazaks, National Institute of Research and Innovation
• Shyam Basak, University of Oxford
• Tiankun Zhou, Diamond Light Source
• William K. Myers, University of Oxford
• Margaret D. Doyle, Lawrence Berkeley National Laboratory
• Philipp S. Simon, Lawrence Berkeley National Laboratory
• Shusaku Hayama, Diamond Light Source
• J. Frederick W. Mosselmans, Diamond Light Source
• Alexander T. Stead, University of Oxford
• Abraham O. Oluwole, University of Oxford
• Malkit Sami, University of Oxford
• Ian Clifton, University of Oxford
• Pierre Aller, Diamond Light Source
• Asmit Bhowmick, Lawrence Berkeley National Laboratory
• Anthony Tumber, University of Oxford
• Jos J.A.G. Kamps, University of Oxford
• Karina Calvopiña, University of Oxford
• Agata Butryn, Diamond Light Source
• Robert Bosman, Diamond Light Source
• Arjun Cheema, University of Oxford
• Jordan Welsh, University of Oxford
• James D.S. Linyard, University of Oxford
• Frederick J.H. Foster, University of Oxford
• Pauline A. Lang, University of Oxford
• Mariska de Munnik, University of Oxford
• Stephen M. Keable, Lawrence Berkeley National Laboratory
• Medhanjali Dasgupta, Lawrence Berkeley National Laboratory

Document Type

Article

Publication Date

10-1-2026

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 O2 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. (Figure presented.)

Publication Title

Nature Catalysis

Recommended Citation

Rabe, P., Makita, H., Southwart, R., Thomas, M., Kazaks, M., Basak, S., Zhou, T., Myers, W., Doyle, M., Simon, P., Hayama, S., Mosselmans, J., Stead, A., Oluwole, A., Sami, M., Clifton, I., Aller, P., Bhowmick, A., Tumber, A., Kamps, J., Calvopiña, K., Butryn, A., Bosman, R., Cheema, A., Welsh, J., Linyard, J., Foster, F., Lang, P., de Munnik, M., Keable, S., & Dasgupta, M. (2026). Unanticipated intermediates during isopenicillin N synthase catalysis identified by time-resolved X-ray free-electron laser studies. Nature Catalysis, 9(10), 1141-1157. http://doi.org/10.1038/s41929-026-01618-4
Retrieved from: https://digitalcommons.mtu.edu/michigantech-p2/3072