Structure of β-lactam synthetase reveals how to synthesize antibiotics instead of asparagine

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Abstract

The enzyme β-lactam synthetase (β-LS) catalyzes the formation of the β-lactam ring in clavulanic acid, a clinically important β-lactamase inhibitor. Whereas the penicillin β-lactam ring is generated by isopenicillin N synthase (IPNS) in the presence of ferrous ion and dioxygen, β-LS uses ATP and Mg2+ as cofactors. According to sequence alignments, β-LS is homologous to class B asparagine synthetases (AS-Bs), ATP/Mg2+-dependent enzymes that convert aspartic acid to asparagine. Here we report the first crystal structure of a β-LS. The 1.95 Å resolution structure of Streptomyces clavuligerus β-LS provides a fully resolved view of the active site in which substrate, closely related ATP analog α,β-methyleneadenosine 5′-triphosphate (AMP-CPP) and a single Mg2+ ion are present. A high degree of substrate preorganization is observed. Comparison to Escherichia coli AS-B reveals the evolutionary changes that have taken place in β-LS that impede interdomain reaction, which is essential in AS-B, and that accommodate β-lactam formation. The structural data provide the opportunity to alter the synthetic potential of β-LS, perhaps leading to the creation of new β-lactamase inhibitors and β-lactam antibiotics.

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Figure 1: Reactions catalyzed by β-LS, IPNS and AS-B.
Figure 2: Comparison of β-LS and AS-B.
Figure 3: Structure-based sequence alignment of β-LS and AS-B.
Figure 4: The active site.

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Acknowledgements

This work was supported by funds from the David and Lucile Packard Foundation to A.C.R., by an NIH grant to C.A.T. and in part by an NIH training grant to M.T.M. The DND-CAT Synchrotron Research Center at the Advanced Photon Source is supported by the E.I. DuPont de Nemours & Co., The Dow Chemical Company, the NSF and the State of Illinois.

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Correspondence to Amy C. Rosenzweig.

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Miller, M., Bachmann, B., Townsend, C. et al. Structure of β-lactam synthetase reveals how to synthesize antibiotics instead of asparagine. Nat Struct Mol Biol 8, 684–689 (2001) doi:10.1038/90394

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