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The structural basis of cephalosporin formation in a mononuclear ferrous enzyme

Nature Structural & Molecular Biology volume 11, pages 95–101 (2004)Cite this article

Abstract

Deacetoxycephalosporin-C synthase (DAOCS) is a mononuclear ferrous enzyme that transforms penicillins into cephalosporins by inserting a carbon atom into the penicillin nucleus. In the first half-reaction, dioxygen and 2-oxoglutarate produce a reactive iron-oxygen species, succinate and CO2. The oxidizing iron species subsequently reacts with penicillin to give cephalosporin and water. Here we describe high-resolution structures for ferrous DAOCS in complex with penicillins, the cephalosporin product, the cosubstrate and the coproduct. Steady-state kinetic data, quantum-chemical calculations and the new structures indicate a reaction sequence in which a 'booby-trapped' oxidizing species is formed. This species is stabilized by the negative charge of succinate on the iron. The binding sites of succinate and penicillin overlap, and when penicillin replaces succinate, it removes the stabilizing charge, eliciting oxidative attack on itself. Requisite groups of penicillin are within 1 Å of the expected position of a ferryl oxygen in the enzyme–penicillin complex.

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Figure 1: The active site region of DAOCS in complex with substrates and products.
Figure 2: Penicillin binds in the same orientation to DAOCS as it does to isopenicillin N synthase (IPNS).
Figure 3: Quantum chemical models for the oxidizing species.
Figure 4: Substrate inhibition of DAOCS with increasing concentrations of penicillin G or 2-oxoglutarate.
Figure 5: A possible mechanism for the ring expansion catalyzed by DAOCS.

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Acknowledgements

We are grateful to C. Schofield and Oxford Centre for Molecular Sciences for discussions and for a supply of DAOC. We thank T. Gunda and L. Eriksson for help. Maxlab, the European Synchrotron Research Facility and the European Molecular Biology Laboratory are acknowledged for beam time and assistance. This work was supported by the European Union Biotechnology Programme and by the Swedish Research Councils.

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Authors and Affiliations

  1. Department of Cellular and Molecular Biology, Molecular Biophysics, Uppsala University, Box 596, Uppsala, S-751 24, Sweden

    Karin Valegård & Janos Hajdu

  2. Department of Molecular Biosciences, Structural Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Box 590, Uppsala, S-751 24, Sweden

    Anke C Terwisscha van Scheltinga, Linda M Öster & Inger Andersson

  3. Centre d'Ingéniere des Protéines, Université de Liège, Insitut de Chimie B6, Liège, 4000, Belgium

    Alain Dubus

  4. Istituto Guido Donegani, PolimeriEuropa, Via Fauser 4, Novara, 28100, Italy

    Graziella Ranghino

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  1. Karin Valegård
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Correspondence to Karin Valegård.

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Valegård, K., van Scheltinga, A., Dubus, A. et al. The structural basis of cephalosporin formation in a mononuclear ferrous enzyme. Nat Struct Mol Biol 11, 95–101 (2004). https://doi.org/10.1038/nsmb712

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