Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723.
A pentachlorophenol (PCP) hydroxylase which catalyzed the conversion of PCP to 2,3,5,6-tetrachlorohydroquinone and released iodide from triiodophenol in the presence of NADPH and oxygen was identified. The enzyme was purified by protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography, gel filtration chromatography, and crystallization. The enzyme was a monomer with a molecular weight of 63,000. Under certain conditions, dimer and multimer conformations were also observed. The pI of the enzyme was pH 4.3. The optimal conditions for activity were a pH of 7.5 to 8.5 and a temperature of 40 degrees C. Each enzyme molecule contained one flavin adenine dinucleotide molecule. The Km for PCP was 30 microM and the Vmax was 16 mumol/min/mg of protein. The enzymatic reaction required 2 mol of NADPH per mol of halogenated substrate. On the basis of the data we present, it is likely that PCP hydroxylase is a flavoprotein monooxygenase. The addition of flavins to the reaction mixture did not stimulate the enzymatic reaction; however, we identified the photodegradation of triiodophenol and tribromophenol, but not PCP, by flavin mononucleotide or riboflavin and light.
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Department of Bacteriology and Biochemistry, University of Idaho, Moscow 83843.
Orser
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D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003460:Crystallization; D005417:Flavobacterium / Q000201:enzymology*; D005420:Flavoproteins / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D013053:Spectrophotometry; D013816:Thermodynamics
D005420:Flavoproteins; D007202:Indicators and Reagents; D006899:Mixed Function Oxygenases; C069781:pentachlorophenol monooxygenase