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

Abstract

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.