Purification and catalytic properties of the chlorophenol 4-monooxygenase from Burkholderia cepacia strain AC1100

• Published in: Biochimica et biophysica acta Vol. 1547; no. 2; pp. 288 - 301
• Main Authors: Martin-Le Garrec, Gaelle, Artaud, Isabelle, Capeillère-Blandin, Chantal
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 11.06.2001
• Subjects: Blast search; Burkholderia cepacia - enzymology; Chlorophenols - chemistry; Enzyme Inhibitors - pharmacology; Flavin monooxygenase; Hydroxylation; Kinetics; Methimazole; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - isolation & purification; Mixed Function Oxygenases - metabolism; Models, Chemical; NAD - chemistry; Oxygen - chemistry; Substrate Specificity; Trichlorophenol monooxygenase; Methimazole; 2,6-DCP, 2,6-dichlorophenol; Trichlorophenol monooxygenase; TCPMO, trichlorophenol monooxygenase(s); MMI, methimazole; PHBH, p-hydroxybenzoate hydroxylase; 2,4,5-TCP, 2,4,5-trichlorophenol; 5-CHQ, 5-chloro-1,2,4-trihydroxybenzene; DTT, dithiothreitol; TeCH, 2,3,4,5-tetrachlorohydroquinone; PMSF, phenylmethylsulfonyl fluoride; Blast search; 2,6-DCHQ, 2,6-dichlorohydroquinone; TeCP, 2,3,4,5-tetrachlorophenol; Flavin monooxygenase; 2,5-DCHQ, 2,5-dichlorohydroquinone; 2,4,5-T, 2,4,5-trichlorophenoxyacetate
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(01)00197-2

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 μM, 32±5 μM and 4±2 μM 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 μM. 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.