Purification and characterization of catechol 2,3-dioxygenase from the aniline degradation pathway of Acinetobacter sp. YAA and its mutant enzyme, which resists substrate inhibition.
Catechol 2,3-dioxygenase (C23O), a key enzyme in the meta-cleavage pathway of catechol metabolism, was purified from cell extract of recombinant Escherichia coli JM109 harboring the C23O gene (atdB) cloned from an aniline-degrading bacterium Acinetobacter sp. YAA. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography analysis suggested that the enzyme (AtdB) has a molecular mass of 35 kDa as a monomer and forms a tetrameric structure. It showed relative meta-cleavage activities for the following catechols tested: catechol (100%), 3-methylcatechol (19%), 4-methylcatechol (57%), 4-chlorocatechol (46%), and 2,3-dihydroxybiphenyl (5%). To elevate the activity, a DNA self-shuffling experiment was carried out using the atdB gene. One mutant enzyme, named AtdBE286K, was obtained. It had one amino acid substitution, E286K, and showed 2.4-fold higher C23O activity than the wild-type enzyme at 100 microM. Kinetic analysis of these enzymes revealed that the wild-type enzyme suffered from substrate inhibition at >2 microM, while the mutant enzyme loosened substrate inhibition.
Takeo
Masahiro
M
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, Himeji, Hyogo, Japan. takeo@eng.u-hyogo.ac.jp
Nishimura
Munehiro
M
Shirai
Mizuho
M
Takahashi
Hana
H
Negoro
Seiji
S
D000150:Acinetobacter / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000378:metabolism*; D050557:Catechol 2,3-Dioxygenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data
D000814:Aniline Compounds; D050557:Catechol 2,3-Dioxygenase; C023650:aniline