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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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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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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DOI: https://doi.org/10.1038/29575
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