Mutational evidence supporting the involvement of tripartite residues His183, Asp185, and His243 in Streptomyces clavuligerus deacetoxycephalosporin C synthase for catalysis.
Deacetoxycephalosporin C synthase (DAOCS) is a non-heme iron-binding and alpha-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.
Sim
J
J
Department of Microbiology, Faculty of Medicine, National University of Singapore, Singapore.
Sim
T S
TS
D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins; D017433:Protein Structure, Secondary; D012995:Solubility; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D046915:Penicillin-Binding Proteins; D001224:Aspartic Acid; D006639:Histidine; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase