C-terminus mutations of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase with improved activity toward penicillin analogs.
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.
Wu
Xiao-Bin
XB
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, PR China.
Fan
Ke-Qiang
KQ
Wang
Qin-Hong
QH
Yang
Ke-Qian
KQ
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*
D010406:Penicillins; D010105:Oxygenases; C045288:deacetoxycephalosporin C hydroxylase