title: Degradation of 3-nitrophenol by Pseudomonas putida B2 occurs via 1,2,4-benzenetriol.
abstract: Growth of Pseudomonas putida B2 in chemostat cultures on a mixture of 3-nitrophenol and glucose induced 3-nitrophenol and 1,2,4-benzenetriol-dependent oxygen uptake activities. Anaerobic incubations of cell suspensions with 3-nitrophenol resulted in complete conversions of the substrate to ammonia and 1,2,4-benzenetriol. This indicates that P. putida B2 degrades 3-nitrophenol via 1,2,4-benzenetriol, via a pathway involving a hydroxylaminolyase. Involvement of this pathway in nitroaromatic metabolism has previously only been found for degradation of 4-nitrobenzoate. Reduction of 3 nitrophenol by cell-free extracts was strictly NADPH-dependent. Attempts to purify the enzymes responsible for 3-nitrophenol metabolism were unsuccessful, because their activities were extremely unstable. 3-Nitrophenol reductase was therefore characterized in cell-free extracts. The enzyme had a sharp pH optimum at pH 7 and a temperature optimum at 25 degrees C. At 30 degrees C, reductase activity was completely destroyed within one hour, while at 0 degrees C, the activity in cell-free extracts was over 100-fold more stable. The Km values for NADPH and 3-nitrophenol were estimated at 0.17 mM and below 2 microM, respectively. The substrate specificity of the reductase activity was very broad: all 17 nitroaromatics tested were reduced by cell-free extracts. However, neither intact cells nor cell-free extracts could convert a set of synthesized hydroxylaminoaromatic compounds to the corresponding catechols and ammonia. Apparently, the hydroxylaminolyase of P. putida B2 has a very narrow substrate specificity, indicating that this organism is not a suitable biocatalyst for the industrial production of catechols from nitroaromatics.
authors.affiliation: Wageningen Agricultural University, Department of Food Science, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Meulenberg
authors.forename: M
authors.initials: M
authors.lastname: Pepi
authors.forename: J A
authors.initials: JA
authors.lastname: de Bont
mesh_terms: D001673:Biodegradation, Environmental; D002396:Catechols / Q000378:metabolism; D002474:Cell-Free System; D003470:Culture Media; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006873:Hydroquinones / Q000378:metabolism*; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism*; D009601:Nitroreductases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D010101:Oxygen Consumption; D016958:Pseudomonas putida / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002396:Catechols; D003470:Culture Media; D004791:Enzyme Inhibitors; D006873:Hydroquinones; D009596:Nitrophenols; C013038:hydroxyhydroquinone; C082064:nitrophenol reductase; D009601:Nitroreductases; C049017:3-nitrophenol