Purification and characterization of chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100.
Burkholderia (formerly Pseudomonas) cepacia AC1100 mineralizes the herbicide 2,4,5-trichlorophenoxyacetate (2,4,5-T), and the first intermediate of 2,4,5-T degradation is 2,4,5-trichlorophenol. Chlorophenol 4-monooxygenase activity responsible for 2,4,5-trichlorophenol degradation was detected in the cell extract. The enzyme consisted of two components separated during purification, and both were purified to more than 95% homogeneity. The reconstituted enzyme catalyzed the hydroxylation of several tested chlorophenols with the coconsumption of NADH and oxygen. In addition to chlorophenols, the enzyme also hydroxylated some chloro-p-hydroquinones with the coconsumption of NADH and oxygen. Apparently, the single enzyme was responsible for converting 2,4,5-trichlorophenol to 2,5-dichloro-p-hydroquinone and then to 5-chlorohydroxyquinol (5-chloro-1,2,4-trihydroxybenzene). Component A had a molecular weight of 22,000 and contained flavin adenine dinucleotide. Component A alone catalyzed NADH-dependent cytochrome c reduction, indicating that it had reductase activity. Component B had a molecular weight of 58,000, and no catalytic activity has yet been shown by itself.
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Department of Microbiology, Washington State University Tri-Cities, Richland 99352, USA.
D016956:Burkholderia cepacia / Q000201:enzymology*; D002733:Chlorophenols / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006900:Hydroxylation; D006912:Hydroxyquinolines / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008956:Models, Chemical; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010101:Oxygen Consumption; D013379:Substrate Specificity
D002733:Chlorophenols; D006912:Hydroxyquinolines; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D006899:Mixed Function Oxygenases; C099366:chlorophenol 4-monooxygenase