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J. Biol. Chem., Vol. 281, Issue 1, 279-287, January 6, 2006

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ORF17 from the Clavulanic Acid Biosynthesis Gene Cluster Catalyzes the ATP-dependent Formation of N-Glycyl-clavaminic Acid^*

Haren Arulanantham, Nadia J. Kershaw, Kirsty S. Hewitson, Claire E. Hughes, Jan E. Thirkettle, and Christopher J. Schofield1

From the Department of Chemistry and The Oxford Centre for Molecular Sciences, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA and GlaxoSmithKline Pharmaceuticals, Clarendon Road, Worthing, West Sussex BN14 8QH, United Kingdom

(3R,5R)-Clavulanic acid, a clinically used inhibitor of serine -lactamases, is produced by fermentation of Streptomyces clavuligerus. The early steps in clavulanic acid biosynthesis leading to the bicyclic -lactam intermediate (3S,5S)-clavaminic acid have been defined. However, the mechanism by which (3S,5S)-clavaminic acid is converted to the penultimate intermediate (3R,5R)-clavaldehyde is unclear. Disruption of orf15 or orf16, of the clavulanic acid biosynthesis gene cluster, blocks clavulanic acid production and leads to the accumulation of N-acetyl-glycyl-clavaminic acid and N-glycyl-clavaminic acid, suggesting that these compounds are intermediates in the pathway. Two alternative start codons have been proposed for orf17 to encode for two possible polypeptides, one of which has 92 N-terminal residues less then the other. The shorter version of orf17 was successfully expressed in Escherichia coli and purified as a monomeric protein. Sequence analyses predicting the ORF17 protein to be a member of the ATP-grasp fold superfamily were supported by soft ionization mass spectrometric analyses that demonstrated binding of ATP to the ORF17 protein. Semisynthetic clavaminic acid, prepared by in vitro reconstitution of the biosynthetic pathway from the synthetically accessible intermediate proclavaminic acid, was shown by mass spectrometric analyses to be converted to N-glycyl-clavaminic acid in the presence of ORF17, ATP, and glycine. Under the same conditions N-acetyl-glycine and clavaminic acid were not converted to N-acetyl-glycyl-clavaminic acid. The specificity of ORF17 as an N-glycyl-clavaminic acid synthetase, together with the reported accumulation of N-glycyl-clavaminic acid in orf15 and orf16 disruption mutants, suggested that N-glycyl-clavaminic acid is an intermediate in clavulanic acid biosynthesis.

Received for publication, July 15, 2005 , and in revised form, October 25, 2005.

^* This work was supported by grants from the Biotechnology and Biological Sciences Research Council, the European Union, the Glasstone Fellowship, and GlaxoSmithKline PLC. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Tel.: 44-1865-275625; Fax: 44-1865-275654; E-mail: christopher.schofield{at}chem.ox.ac.uk.

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