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The reaction cycle of isopenicillin N synthase observed by X-ray diffraction

Nature volume 401, pages 721–724 (1999)Cite this article

Abstract

Isopenicillin N synthase (IPNS), a non-haem iron-dependent oxidase, catalyses the biosynthesis of isopenicillin N (IPN), the precursor of all penicillins and cephalosporins1. The key steps in this reaction are the two iron-dioxygen-mediated ring closures of the tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV). It has been proposed that the four-membered β-lactam ring forms initially, associated with a highly oxidized iron(IV)-oxo (ferryl) moiety, which subsequently mediates closure of the five-membered thiazolidine ring2. Here we describe observation of the IPNS reaction in crystals by X-ray crystallography. IPNS·Fe2+·substrate crystals were grown anaerobically3,4, exposed to high pressures of oxygen to promote reaction and frozen, and their structures were elucidated by X-ray diffraction. Using the natural substrate ACV, this resulted in the IPNS·Fe2+·IPN product complex. With the substrate analogue, δ-(L-α-aminoadipoyl)-L-cysteinyl-L-S-methylcysteine (ACmC) in the crystal, the reaction cycle was interrupted at the monocyclic stage. These mono- and bicyclic structures support our hypothesis of a two-stage reaction sequence leading to penicillin. Furthermore, the formation of a monocyclic sulphoxide product from ACmC is most simply explained by the interception of a high-valency iron-oxo species.

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Figure 1: Structures showing changes in the active-site region.
Figure 2: Proposed mechanisms for the oxidation of ACV and ACmC to bicyclic and monocyclic products, respectively.
Figure 3: Stereo views of the two substrates and two products overlaid.
Figure 4: The β-lactam ring is stabilized against fragmentation by hydrogen bonds from well-ordered water molecules observed in both the product structures.

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Acknowledgements

We thank K. Harlos, C. Schofield, A. Long, J. Elkins, J. Ogle, P. Wright, R. Wilmouth, S. Lee, A. Salmeen, J. Pitt, J. Keeping and the scientists at SRS Daresbury, EMBL Hamburg and ESRF Grenoble for help and discussions. Financial support was provided by the MRC, BBSRC and ESPRC. P.L.R. thanks the Royal Society for financial support. N.I.B. was supported by a German DAAD fellowship. P.J.R. thanks the Rhodes Trust for support.

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  1. Nicolai I. Burzlaff, Peter J. Rutledge and Michael Pickford: These authors contributed equally to this work

Authors and Affiliations

  1. The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QY, UK

    Nicolai I. Burzlaff, Peter J. Rutledge, Ian J. Clifton, Charles M. H. Hensgens, Robert M. Adlington, Peter L. Roach & Jack E. Baldwin

  2. The Laboratory of Molecular Biophysics, University of Oxford, South Parks Road, Oxford, OX1 3QY, UK

    Michael Pickford

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Correspondence to Jack E. Baldwin.

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Burzlaff, N., Rutledge, P., Clifton, I. et al. The reaction cycle of isopenicillin N synthase observed by X-ray diffraction. Nature 401, 721–724 (1999). https://doi.org/10.1038/44400

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