The kinetic properties of various R258 mutants of deacetoxycephalosporin C synthase. |
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| Authors: | Hwei-Jen Lee Young-Fung Dai Chia-Yang Shiau Christopher J Schofield Matthew D Lloyd |
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| Affiliation: | Department of Biochemistry and Institute of Medical Science, National Defense Medical Centre, Taipei, Taiwan, ROC. |
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| 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. |
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