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J. Biol. Chem., Vol. 279, Issue 15, 15420-15426, April 9, 2004

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Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase^*

Matthew D. Lloyd¶, Sarah J. Lipscomb||, Kirsty S. Hewitson||, Charles M. H. Hensgens||, Jack E. Baldwin||, and Christopher J. Schofield||

From the Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom and ^||Department of Chemistry and Oxford Centre for Molecular Sciences, Mansfield Road, Oxford OX1 3TA, United Kingdom

Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (310 mutant) enhanced ring-expansion of penicillin G by 2-fold. A double mutant, 310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The 310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K_m = 2.00 ± 0.47 compared with 6.02 ± 0.97 mM for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.

Received for publication, December 19, 2003 , and in revised form, January 16, 2004.

^* This work was supported by the BBSRC, EPSRC, MRC, Wellcome Trust, and EU. 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.

Both authors contributed equally to this work.

^¶ To whom correspondence should be addressed. Fax: 44-1225-386114; E-mail: M.D.Lloyd{at}bath.ac.uk.

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