Alteration of the co-substrate selectivity of deacetoxycephalosporin C synthase: The role of arginine 258

Hwei-Jen Lee, Matthew D. Lloyd, Ian J. Clifton, Karl Harlos, Alain Dubus, Jack E. Baldwin, Jean Marie Frere, Christopher J. Schofield

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36 Citations (SciVal)


Deacetoxycephalosporin C synthase is an iron(II) 2-oxoglutaratedependent oxygenase that catalyzes the oxidative ring-expansion of penicillin N to deacetoxycephalosporin C. The wild-type enzyme is only able to efficiently utilize 2-oxoglutarate and 2-oxoadipate as a 2-oxoacid co-substrate. Mutation of arginine 258, the side chain of which forms an electrostatic interaction with the 5-carboxylate of the 2-oxoglutarate co-substrate, to a glutamine residue reduced activity to about 5% of the wild-type enzyme with 2-oxoglutarate. However, other aliphatic 2-oxoacids, which were not cosubstrates for the wild-type enzyme, were utilized by the R258Q mutant. These 2-oxoacids "rescued" catalytic activity to the level observed for the wild-type enzyme as judged by penicillin N and G conversion. These cosubstrates underwent oxidative decarboxylation as observed for 2-oxoglutarate in the normal reaction with the wild-type enzyme. Crystal structures of the iron(II)-2-oxo-3-methylbutanoate (1.5 Å), and iron(II)-2-oxo-4-methylpentanoate (1.6 Å) enzyme complexes were obtained, which reveal the molecular basis for this "chemical co-substrate rescue" and help to rationalize the co-substrate selectivity of 2-oxoglutaratedependent oxygenases.

Original languageEnglish
Pages (from-to)18290-18295
Number of pages6
JournalJournal of Biological Chemistry
Issue number21
Publication statusPublished - 25 Jan 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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