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...

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Bibliographic Details
Published in:FEMS microbiology letters 2005-05, Vol.246 (1), p.103-110
Main Authors: Wu, Xiao-Bin, Fan, Ke-Qiang, Wang, Qin-Hong, Yang, Ke-Qian
Format: Article
Language:English
Subjects:
Acremonium - enzymology
Acremonium - genetics
Acremonium chrysogenum
Amino Acid Substitution
Analogs
Antibiotics
Bacteriology
Biological and medical sciences
C-Terminus
DAC
DAOC
Deacetoxy/deacetylcephalosporin C synthase
Enzymes
Fundamental and applied biological sciences. Psychology
Genetics
Hydroxylation
Kinetics
Leucine
Microbiology
Models, Molecular
Mutagenesis
Mutagenesis, Site-Directed
Mutation
Oxygenases - chemistry
Oxygenases - genetics
Oxygenases - isolation & purification
Oxygenases - metabolism
Penicillin
Penicillin N
Penicillins - metabolism
Substrates
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Summary: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.
ISSN:0378-1097
1574-6968
DOI:10.1016/j.femsle.2005.03.043