title: Relevant double mutations in bioengineered Streptomyces clavuligerus deacetoxycephalosporin C synthase result in higher binding specificities which improve penicillin bioconversion.
abstract: Streptomyces clavuligerus deacetoxycephalosporin C synthase (ScDAOCS) is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid, which is a precursor for cephalosporin synthesis. Single mutations of six amino acid residues, V275, C281, N304, I305, R306, and R307, were previously shown to result in enhanced levels of ampicillin conversion, with activities ranging from 129 to 346% of the wild-type activity. In this study, these mutations were paired to investigate their effects on enzyme catalysis. The bioassay results showed that the C-terminal mutations (N304X [where X is alanine, leucine, methionine, lysine, or arginine], I305M, R306L, and R307L) in combination with C281Y substantially increased the conversion of ampicillin; the activity was up to 491% of the wild-type activity. Similar improvements were observed for converting carbenicillin (up to 1,347% of the wild-type activity) and phenethicillin (up to 1,109% of the wild-type activity). Interestingly, the N304X R306L double mutants exhibited lower activities for penicillin G conversion, and activities that were 40 to 114% of wild-type enzyme activity were detected. Based on kinetic studies using ampicillin, it was clear that the increases in the activities of the double mutants relative to those of the corresponding single mutants were due to enhanced substrate binding affinities. These results also validated the finding that the N304R and I305M mutations are ideal for increasing the substrate binding affinity and turnover rate of the enzyme, respectively. This study provided further insight into the structure-function interaction of ScDAOCS with different penicillin substrates, thus providing a useful platform for further rational modification of its enzymatic properties.
authors: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore.
authors: Kian Sim
authors: KS
authors: Goo
authors: Chun Song
authors: CS
authors: Chua
authors: Tiow-Suan
authors: TS
authors: Sim
mesh_terms: D000667:Ampicillin / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D019751:Intramolecular Transferases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D046915:Penicillin-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D010406:Penicillins / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D000667:Ampicillin; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase