title: New strategy of site-directed mutagenesis identifies new sites to improve Streptomyces clavuligerus deacetoxycephalosporin C synthase activity toward penicillin G.
abstract: Based on multiple sequence alignment of different deacetoxycephalosporin C synthase (DAOCSs) and the crystal structure of Streptomyces clavuligerus DAOCS, 2-oxoglutarate, and penicillin G triple complex, ten residues (Y184, V245, S261, C37, T42, V51, S59, A61, Q126, and T213) not directly involved in substrate recognition were selected as mutational targets. Twenty one mutants were generated and characterized, and five (Q126M, T213V, S261M, S261A, and Y184A) showed improved activity toward penicillin G, with 1.45- to 4.50-fold increment in the k                         (cat)/K                         (m). Q126, T213, and S261 are identified for the first time, as sites with significant effect on enzyme activity.
authors: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing, People's Republic of China.
authors: Junjie
authors: J
authors: Ji
authors: Xiuyun
authors: X
authors: Tian
authors: Keqiang
authors: K
authors: Fan
authors: Keqian
authors: K
authors: Yang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010400:Penicillin G / Q000378:metabolism*; D046915:Penicillin-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D046915:Penicillin-Binding Proteins; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase; D010400:Penicillin G