title: Purification and characterization of meta-cleavage compound hydrolase from a carbazole degrader Pseudomonas resinovorans strain CA10.
abstract: 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.
authors.affiliation: Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nojiri
authors.forename: Hiroko
authors.initials: H
authors.lastname: Taira
authors.forename: Kenichi
authors.initials: K
authors.lastname: Iwata
authors.forename: Kenichi
authors.initials: K
authors.lastname: Morii
authors.forename: Jeong-Won
authors.initials: JW
authors.lastname: Nam
authors.forename: Takako
authors.initials: T
authors.lastname: Yoshida
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Habe
authors.forename: Shugo
authors.initials: S
authors.lastname: Nakamura
authors.forename: Kentaro
authors.initials: K
authors.lastname: Shimizu
authors.forename: Hisakazu
authors.initials: H
authors.lastname: Yamane
authors.forename: Toshio
authors.initials: T
authors.lastname: Omori
mesh_terms: 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
chemical_list: D002227:Carbazoles; D003470:Culture Media; D004269:DNA, Bacterial; C476804:2-hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4-dienoic acid hydrolase; D006867:Hydrolases