Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/130876
Title: Mutational evidence supporting the involvement of tripartite residues his 183, asp 185, and his243 in Streptomyces clavuligerus deacetoxycephalosporin C synthase for catalysis
Authors: Sim, J. 
Sim, T.-S. 
Keywords: Deacetoxycephalosporin C synthase
Histidine
Iron-binding
Site-directed mutagenesis
Streptomyces clavuligerus
Issue Date: Apr-2000
Source: Sim, J., Sim, T.-S. (2000-04). Mutational evidence supporting the involvement of tripartite residues his 183, asp 185, and his243 in Streptomyces clavuligerus deacetoxycephalosporin C synthase for catalysis. Bioscience, Biotechnology and Biochemistry 64 (4) : 828-832. ScholarBank@NUS Repository.
Abstract: Deacetoxycephalosporin C synthase (DAOCS) is a non-heme iron-binding and α-ketoglutarate dependent enzyme involved in catalyzing the biosynthesis of cephalosporins and cephamycins, antibiotics more potent than penicillins. In the crystal structure complex of Streptomyces clavuligerus DAOCS (scDAOCS), it was proposed that histidine-183, aspartate-185, and histidine-243 are putative iron-binding ligands. However, coordinates proposed for crystal structures of proteins may not definitely comply with catalysis. Hence, site-directed mutagenesis was done to replace each of these amino acid residues with leucine. The constructed expression vectors bearing the mutations were found to express the respective scDAOCS mutant enzymes at high levels in Escherichia coli BL21(DE3). Through enzymatic assays, it was shown that while the wildtype enzyme could convert penicillin to a more active cephalosporin, the substitution of the three proposed iron-binding sites of scDAOCS completely abolished the same activity in the respective mutant enzymes. Thus, these results clearly indicate that histidine-183, aspartate-185, and histidine-243 of scDAOCS are essential for the ring expansion activity.
Source Title: Bioscience, Biotechnology and Biochemistry
URI: http://scholarbank.nus.edu.sg/handle/10635/130876
ISSN: 09168451
Appears in Collections:Staff Publications

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