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C-terminus mutations of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase with improved activity toward penicillin analogs
Authors:Wu Xiao-Bin  Fan Ke-Qiang  Wang Qin-Hong  Yang Ke-Qian
Institution:State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, PR China.
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.
Keywords:DAOC  DAC  Deacetoxy/deacetylcephalosporin C synthase  C-terminus  Mutagenesis              Acremonium chrysogenum            Kinetics
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