title: The kinetic properties of various R258 mutants of deacetoxycephalosporin C synthase.
abstract: 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.affiliation: Department of Biochemistry and Institute of Medical Science, National Defense Medical Centre, Taipei, Taiwan, ROC.
authors.forename: Hwei-Jen
authors.initials: HJ
authors.lastname: Lee
authors.forename: Young-Fung
authors.initials: YF
authors.lastname: Dai
authors.forename: Chia-Yang
authors.initials: CY
authors.lastname: Shiau
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Lloyd
mesh_terms: D000667:Ampicillin / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D007656:Ketoglutaric Acids; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D000667:Ampicillin; D001120:Arginine; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase