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Site-directedmutagenesis was used to investigate the control of 2-oxoacid cosubstrate selectivity by deacetoxycephalo-sporin C synthase. The wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids (e.g. 2-oxohexanoic acid, 2-oxo-4-methyl-penta-noic acid) as the cosubstrate. The followingmutant enzymes were produced: R258A, R258L, R258F, R258H and R258K. All of the mutants have broadened cosubstrate selectivity and were able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization by all mutants was decreased as compared to the wild-type enzyme, and in some cases activity was abolished with the natural cosubstrate | Eur. J. Biochem. 270 1301-1307 2003 FEBS 2003 doi 10.1046 j.1432-1033.2003.03500.x The kinetic properties of various R258 mutants of deacetoxycephalosporin C synthase Hwei-Jen Lee1 Young-Fung Dai1 Chia-Yang Shiau2 Christopher J. Schofield3 and Matthew D. Lloyd4 1Department of Biochemistry National Defense Medical Centre Taipei Taiwan ROC 2Institute of Medical Science National Defense Medical Centre Taipei Taiwan ROC 3 The Oxford Centre for Molecular Sciences and Dyson Perrins Laboratory South Parks Road Oxford UK 4Department of Pharmacy and Pharmacology University of Bath Claverton Down Bath UK Site-directed mutagenesis was used to investigate the control of 2-oxoacid cosubstrate selectivity by deacetoxycephalosporin C synthase. The wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids e.g. 2-oxohexanoic acid 2-oxo-4-methyl-penta-noic acid as the cosubstrate. The following mutant enzymes were produced R258A R258L R258F R258H and R258K. All of the mutants have broadened cosubstrate selectivity and were able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization by all mutants was decreased as compared to the wild-type enzyme and in some cases activity was abolished with the natural cosubstrate. Keywords b-lactam biosynthesis cephem chemical cosubstrate rescue nonhaem iron II oxygenase 2-oxoglutarate. Deacetoxycephalosporin C synthase DAOCS Swiss-Prot P18548 catalyses a key step in the cephamycin C biosynthetic pathway in Streptomyces clavuligerus i.e. the ringexpansion of penicillin N 1 to deacetoxycephalosporin C DAOC 2 1-7 Scheme 1 . A sequence-related oxygenase deacetylcephalosporin C synthase DACS Swiss-Prot 42220 catalyses the subsequent hydroxylation of the exocy-clic methyl group of 2 to give deacetylcephalosporin C DAC 3 . The DAC product is then converted by a series of reactions including 7-hydroxylation into cephamycin C 4 . Oxidative reactions in this pathway are catalysed by .
