Purification and characterization of meta-cleavage compound hydrolase from a carbazole degrader Pseudomonas resinovorans strain CA10.
2-Hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4dienoic acid [6-(2'-aminophenyl)-HODA] hydrolase, involved in carbazole degradation by Pseudomonas resinovorans strain CA10, was purified to near homogeneity from an overexpressing Escherichia coli strain. The enzyme was dimeric, and its optimum pH was 7.0-7.5. Phylogenetic analysis showed the close relationship of this enzyme to other hydrolases involved in the degradation of monocyclic aromatic compounds, and this enzyme was specific for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (6-phenyl-HODA), having little activity toward 2-hydroxy-6-oxohepta-2,4-dienoic acid and 2-hydroxymuconic semialdehyde. The enzyme had a Km of 2.51 microM and k(cat) of 2.14 (s(-1)) for 6-phenyl-HODA (50 mM sodium phosphate, pH 7.5, 25 degrees C). The effect of the presence of an amino group or hydroxyl group at the 2'-position of phenyl moiety of 6-phenyl-HODA on the enzyme activity was found to be small; the activity decreased only in the order of 6-(2'-aminophenyl)-HODA (2.44 U/mg) > 6-phenyl-HODA (1.99 U / mg) > 2-hydroxy-6-oxo-6-(2'-hydroxyphenyl)-hexa-2,4-dienoic acid (1.05 U/mg). The effects of 2'-substitution on the activity were in accordance with the predicted reactivity based on the calculated lowest unoccupied molecular orbital energy for these substrates.
Nojiri
Hideaki
H
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Taira
Hiroko
H
Iwata
Kenichi
K
Morii
Kenichi
K
Nam
Jeong-Won
JW
Yoshida
Takako
T
Habe
Hiroshi
H
Nakamura
Shugo
S
Shimizu
Kentaro
K
Yamane
Hisakazu
H
Omori
Toshio
T
D001711:Biotransformation; D002227:Carbazoles / Q000378:metabolism*; D003470:Culture Media; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D010802:Phylogeny; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature
D002227:Carbazoles; D003470:Culture Media; D004269:DNA, Bacterial; C476804:2-hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4-dienoic acid hydrolase; D006867:Hydrolases