title: C-terminus mutations of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase with improved activity toward penicillin analogs.
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 (DAOC) and the hydroxylation of the latter to deacetylcephalosporin C (DAC). Three residues N305, R307 and R308 located in close proximity to the C-terminus of acDAOC/DACS were each mutated to leucine. The N305L and R308L mutant acDAOC/DACSs showed significant improvement in their ability to convert penicillin analogs. R308 was identified for the first time as a critical residue for DAOC/DACS activity. Kinetic analyses of purified R308L enzyme indicated its improved catalytic efficiency is due to combined improvements of K(m) and k(cat). Comparative modeling of acDAOC/DACS supports the involvement of R308 in the formation of substrate-binding pocket.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, PR China.
authors.forename: Xiao-Bin
authors.initials: XB
authors.lastname: Wu
authors.forename: Ke-Qiang
authors.initials: KQ
authors.lastname: Fan
authors.forename: Qin-Hong
authors.initials: QH
authors.lastname: Wang
authors.forename: Ke-Qian
authors.initials: KQ
authors.lastname: Yang
mesh_terms: D000164:Acremonium / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010406:Penicillins / Q000378:metabolism*
chemical_list: D010406:Penicillins; D010105:Oxygenases; C045288:deacetoxycephalosporin C hydroxylase