The kinetic properties of various R258 mutants of deacetoxycephalosporin C synthase.
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.
Lee
Hwei-Jen
HJ
Department of Biochemistry and Institute of Medical Science, National Defense Medical Centre, Taipei, Taiwan, ROC.
Dai
Young-Fung
YF
Shiau
Chia-Yang
CY
Schofield
Christopher J
CJ
Lloyd
Matthew D
MD
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
D007656:Ketoglutaric Acids; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D000667:Ampicillin; D001120:Arginine; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase