Authors
Patrick Rabe, Hiroki Makita, Rebecca Southwart, Midhun George Thomas, Mihails Kazaks, Shyam Basak, Tiankun Zhou, William K Myers, Margaret D Doyle, Philipp S Simon, Shusaku Hayama, J Frederick W Mosselmans, Alexander T Stead, Abraham O Oluwole, Malkit Sami, Ian Clifton, Pierre Aller, Asmit Bhowmick, Anthony Tumber, Jos J A G Kamps, Karina Calvopiña, Agata Butryn, Robert Bosman, Arjun Cheema, Jordan Welsh, James D S Linyard, Frederick J H Foster, Pauline A Lang, Mariska de Munnik, Stephen M Keable, Medhanjali Dasgupta, In-Sik Kim, Isabel Bogacz, Kuntal Chatterjee, Kyle D Sutherlin, Corey J Kaminsky, Jürgen Brem, Michael A McDonough, Christina Redfield, James R D Montgomery, Coral Mycroft, Anastasya Shilova, Nicholas Devenish, Frederic P Poitevin, Sebastian Dehe, Vandana Tiwari, Humberto Sanchez, Daniel J Rosenberg, Mark S Hunter, Leland B Gee, Uwe Bergmann, Roberto Alonso-Mori, Jaehyun Park, Shigeki Owada, Kensuke Tono, Danny Axford, Robin L Owen, Daniel W Paley, Nicholas K Sauter, David Mittan-Moreau, Aaron S Brewster, Carol V Robinson, Vittal K Yachandra, Junko Yano, Edgars Suna, Christo Z Christov, Allen M Orville, Jan F Kern, Christopher J Schofield
Published in
Nature catalysis. Volume 9. Issue 10. Pages 1141-1157. Epub Oct 09, 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.
PMID:
42920546
Bibliographic data and abstract were imported from PubMed on 24 Oct 2026.
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