The kinetic properties of various R258 mutants of deacetoxycephalosporin C synthase.

Journal article

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-pentanoic 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.

Authors: Lee, H; Dai, Y; Shiau, C; Schofield, C; Lloyd, MD

• Publication status: Published
• Journal: European journal of biochemistry / FEBS
• Volume: 270
• Issue: 6
• Pages: 1301-1307
• Publication date: 2003-03-01
• DOI: 10.1046/j.1432-1033.2003.03500.x
• EISSN: 1432-1033
• ISSN: 0014-2956
• Language: English
• Keywords: Molecular Structure; Penicillin-Binding Proteins; Mutagenesis, Site-Directed; Models, Molecular; Ketoglutaric Acids; Oxidation-Reduction; Ampicillin; Substrate Specificity; Arginine; Intramolecular Transferases; Penicillins