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Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase

Abstract

Clavaminate synthase (CAS), a remarkable Fe(II)/2-oxoglutarate oxygenase, catalyzes three separate oxidative reactions in the biosynthesis of clavulanic acid, a clinically used inhibitor of serine β-lactamases. The first CAS-catalyzed step (hydroxylation) is separated from the latter two (oxidative cyclization/desaturation) by the action of an amidinohydrolase. Here, we describe crystal structures of CAS in complex with Fe(II), 2-oxoglutarate (2OG) and substrates (N-α-acetyl-L-arginine and proclavaminic acid). They reveal how CAS catalyzes formation of the clavam nucleus, via a process unprecedented in synthetic organic chemistry, and suggest how it discriminates between substrates and controls reaction of its highly reactive ferryl intermediate. The presence of an unpredicted jelly roll β-barrel core in CAS implies divergent evolution within the family of 2OG and related oxygenases. Comparison with other non-heme oxidases/oxygenases reveals flexibility in the position which dioxygen ligates to the iron, in contrast to the analogous heme-using enzymes.

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Figure 1: β-Lactam antibiotics (resistance and biosynthesis) and 2-oxoglutarate dioxygenases.
Figure 2: The role of CAS in clavulanic acid biosynthesis.
Figure 3: The structure of CAS.
Figure 4: Active site of CAS.

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Acknowledgements

We thank H. McNaughton for purification of proclavaminic acid, M. Groves and I. Clifton for help with computing, S. Lee for photography, D. I. Stuart for encouragement, the staff at SRS Daresbury for technical support, our colleagues for encouragement, and the EC, BBSRC, EPSRC and the MRC for financial support.

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Correspondence to Karl Harlos or Christopher J. Schofield.

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Zhang, Z., Ren, J., Stammers, D. et al. Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase. Nat Struct Mol Biol 7, 127–133 (2000). https://doi.org/10.1038/72398

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