title: Purification and characterization of a bacterial nitrophenol oxygenase which converts ortho-nitrophenol to catechol and nitrite.
abstract: A nitrophenol oxygenase which stoichiometrically converted ortho-nitrophenol (ONP) to catechol and nitrite was isolated from Pseudomonas putida B2 and purified. The substrate specificity of the enzyme was broad and included several halogen- and alkyl-substituted ONPs. The oxygenase consisted of a single polypeptide chain with a molecular weight of 58,000 (determined by gel filtration) or 65,000 (determined on a sodium dodecyl sulfate-polyacrylamide gel). The enzymatic reaction was NADPH dependent, and one molecule of oxygen was consumed per molecule of ONP converted. Enzymatic activity was stimulated by magnesium or manganese ions, whereas the addition of flavin adenine dinucleotide, flavin mononucleotide, or reducing agents had no effect. The apparent Kms for ONP and NADPH were 8 and 140 microM, respectively. 2,4-Dinitrophenol competitively (Ki = 0.5 microM) inhibited ONP turnover. The optimal pH for enzyme stability and activity was in the range of 7.5 to 8.0. At 40 degrees C, the enzyme was totally inactivated within 2 min; however, in the presence of 1 mM ONP, 40% of the activity was recovered, even after 10 min. Enzymatic activity was best preserved at -20 degrees C in the presence of 50% glycerol.
authors.affiliation: Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), Basel.
authors.forename: J
authors.initials: J
authors.lastname: Zeyer
authors.forename: H P
authors.initials: HP
authors.lastname: Kocher
mesh_terms: D002384:Catalysis; D002396:Catechols / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009573:Nitrites / Q000378:metabolism*; D009596:Nitrophenols / Q000378:metabolism*; D010105:Oxygenases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D014461:Ultracentrifugation
chemical_list: D002396:Catechols; D009573:Nitrites; D009596:Nitrophenols; C045573:2-nitrophenol; D010105:Oxygenases; C055456:2-nitrophenol oxygenase