title: Saturation mutagenesis of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase R308 site confirms its role in controlling substrate specificity.
abstract: Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C and the hydroxylation of the latter to deacetylcephalosporin C. The R308 residue located in close proximity to the C-terminus of acDAOC/DACS was mutated to the other 19 amino acids. In the resulting mutant pool, R308L, R308I, R308T and R308V showed significant improvement in their ability to convert penicillin analogs, thus confirming the role of R308 in controlling substrate selectivity, the four amino acids all possess short aliphatic sidechains that may improve hydrophobic interactions with the substrates. The mutant R308I showed the highest reactivity for penicillin G, with 3-fold increase in k(cat)/K(m) ratio and 7-fold increase in relative activity.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing, People's Republic of China.
authors.forename: Xiao-Bin
authors.initials: XB
authors.lastname: Wu
authors.forename: Xiu-Yun
authors.initials: XY
authors.lastname: Tian
authors.forename: Jun-Jie
authors.initials: JJ
authors.lastname: Ji
authors.forename: Wei-Bin
authors.initials: WB
authors.lastname: Wu
authors.forename: Ke-Qiang
authors.initials: KQ
authors.lastname: Fan
authors.forename: Ke-Qian
authors.initials: KQ
authors.lastname: Yang
mesh_terms: D000164:Acremonium / Q000201:enzymology* / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D016296:Mutagenesis; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics*; D010400:Penicillin G / Q000031:analogs & derivatives / Q000737:chemistry; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D010088:Oxidoreductases; C041342:desacetoxycephalosporin C synthetase; D010400:Penicillin G