C-terminus modification of Streptomyces clavuligerus deacetoxycephalosporin C synthase improves catalysis with an expanded substrate specificity.
The biosynthesis of cephalosporins is catalyzed by deacetoxycephalosporin C synthase (DAOCS). Based on computational, biochemical, and structural analyses, it has been proposed that modification of the C-terminus of DAOCS might be a constructive strategy for engineering improvement in enzyme activity. Therefore, five hydrophilic residues namely N301, Y302, N304, R306, and R307 located in proximity to the C-terminus of Streptomyces clavuligerus DAOCS (scDAOCS) were selected and each substituted with a hydrophobic leucine residue. Substitutions at positions 304, 306, and 307 created mutant scDAOCSs with improved efficiencies in penicillin analog conversion up to 397%. And since it has been previously advocated that the C-terminus is crucial for guiding substrate entry, a truncated mutant DAOCS was constructed to assess its involvement. The truncation of the C-terminus at position 310 in the wild-type scDAOCS resulted in reduction of indiscriminate conversion of penicillin analog but this defect was compensated by the replacement of asparagine with leucine at position 304.
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Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Republic of Singapore.
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D019943:Amino Acid Substitution; D001681:Biological Assay; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D057927:Hydrophobic and Hydrophilic Interactions; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
D046915:Penicillin-Binding Proteins; D010406:Penicillins; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase