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Structure of a cephalosporin synthase

Nature volume 394, pages 805–809 (1998)Cite this article

Abstract

Penicillins and cephalosporins are among the most widely used therapeutic agents. These antibiotics are produced from fermentation-derived materials as their chemical synthesis is not commercially viable. Unconventional steps in their biosynthesis are catalysed by Fe(II)-dependent oxidases/oxygenases; isopenicillin N synthase (IPNS)1,2 creates in one step the bicyclic nucleus of penicillins, and deacetoxycephalosporin C synthase (DAOCS) catalyses the expansion of the penicillin nucleus into the nucleus of cephalosporins. Both enzymes use dioxygen-derived ferryl intermediates in catalysis but, in contrast to IPNS, the ferryl form of DAOCS is produced by the oxidative splitting of a co-substrate, 2-oxoglutarate (α-ketoglutarate). This route of controlled ferryl formation and reaction is common to many mononuclear ferrous enzymes3, which participate in a broader range of reactions than their well-characterized counterparts, the haem enzymes. Here we report the first crystal structure of a 2-oxoacid-dependent oxygenase. High-resolution structures for apo-DAOCS, the enzyme complexed with Fe(II), and with Fe(II) and 2-oxoglutarate, were obtained from merohedrally twinned crystals. Using a model based on these structures, we propose a mechanism for ferryl formation.

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Figure 1
Figure 2: Comparison of the structures of deacetoxycephalosporin C synthase (DAOCS) and isopenicillin N synthase (IPNS).
Figure 3: Structure of the active site in DAOCS.
Figure 4: Scheme to show the proposed mechanism for the formation of the ferryl intermediate in DAOCS and related 2-oxoacid-dependent ferrous enzymes.

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Acknowledgements

We thank Å. Danielsson and R. Bhikhabhai (Pharmacia Biotech, Uppsala) for improved protein purification procedures; G. Larsson and A. M. Valegård for bigger and better crystals; D.van der Spoel, C. M. R. Wouts, E. Wikman and G. Kleywegt for discussion; C. Andersson for in-house X-ray facilities; V. Biou, Z.-H. Zhang, P. L. Roach, J. Keeping and J. Pitt for their help, and Y. Cerelius and A. Svensson for data collection facilities, at MAX-Lab in Lund; and A. Dahl for a stimulating environment. This work was supported by EU-BIOTECH and the Swedish Research Councils, MFR and NFR. The Oxford Centre for Molecular Sciences is supported by BBSRC and MRC.

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Author notes

  1. Karin Valegård, Anke C. Terwisscha van Scheltinga, Anastassis Perrakis and Andy Thompson: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Biochemistry, Uppsala University, Box 576, S-751 23, Uppsala, Sweden

    Karin Valegård, Takane Hara & Janos Hajdu

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

    Anke C. Terwisscha van Scheltinga, S. Ramaswamy & Inger Andersson

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

    Matthew D. Lloyd, Hwei-Jen Lee, Jack E. Baldwin & Christopher J. Schofield

  4. EMBL Grenoble, c/o ILL, Avenue des Martyrs, BP 156, F-38042 Cedex 9, Grenoble, France

    Anastassis Perrakis & Andy Thompson

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Correspondence to Christopher J. Schofield or Inger Andersson.

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Valegård, K., van Scheltinga, A., Lloyd, M. et al. Structure of a cephalosporin synthase. Nature 394, 805–809 (1998). https://doi.org/10.1038/29575

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