Purification and catalytic properties of the chlorophenol 4-monooxygenase from Burkholderia cepacia strain AC1100.
Burkholderia cepacia strain AC1100 can be induced for the degradation of 2,4,5-trichlorophenol (2,4,5-TCP). We have purified the active enzyme 30-fold to apparent homogeneity with a 44% yield by a two-step chromatographic procedure, and showed that it consists of a single type of subunit of 59 kDa based on SDS-PAGE using Coomassie blue and Sypro staining. This enzyme has no bound prosthetic group but requires exogenous addition of FAD and NADH to perform the dioxygen-dependent hydroxylation in the 4-position of 2,4,6-TCP. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of dioxygen per mol of 2,4,6-TCP with identification of the reaction product as 2,6-dichlorohydroquinone. Steady state kinetic parameters for cofactors and a variety of substrates were determined. Low K(m) values of 1+/-0.1 microM, 32+/-5 microM and 4+/-2 microM were found for FAD, NADH and 2,6-dichlorophenol (2,6-DCP), respectively, under saturating conditions for the two others. In the presence of 2,6-DCP as a substrate, methimazole (MMI) inhibited the enzyme competitively with a K(i)=27 microM. When other polychlorinated substrates were studied, IC(50) values for MMI were found in a range compatible with their apparent affinity. On the basis of aromatic product formation, NADH and O(2) consumption schemes for 2,4,6-TCP and 2,4,5-TCP degradation are discussed. A Blast search revealed that this enzyme has a high sequence identity (60%) with 2,4,6-TCP-4-monooxygenases from Burkholderia pickettii and from Azotobacter sp. strain GP1 which all of them catalyze para hydroxylative dehalogenation.
Martin-Le Garrec
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Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS UMR 8601, Université René Descartes, Paris V, 45 rue des Saints Pères, 75270 Cedex 06, Paris, France.
Artaud
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Capeillère-Blandin
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D016956:Burkholderia cepacia / Q000201:enzymology*; D002733:Chlorophenols / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008956:Models, Chemical; D009243:NAD / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D013379:Substrate Specificity
D002733:Chlorophenols; D004791:Enzyme Inhibitors; D009243:NAD; D006899:Mixed Function Oxygenases; C099366:chlorophenol 4-monooxygenase; C116190:3,5-dichlorophenol; D010100:Oxygen