Martin-Le Garrec G, Artaud I, Capeillère-Blandin C
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.
Biochim Biophys Acta. 2001 Jun 11;1547(2):288-301. 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 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.
洋葱伯克霍尔德菌AC1100菌株可被诱导降解2,4,5-三氯苯酚（2,4,5-TCP）。我们通过两步色谱法将活性酶纯化了30倍，达到表观均一性，产率为44%。基于考马斯亮蓝和Sypro染色的SDS-PAGE分析表明，该酶由一种59 kDa的单一亚基组成。这种酶没有结合辅基，但需要外源添加FAD和NADH才能对2,4,6-TCP的4位进行双氧依赖性羟基化反应。化学计量学研究表明，每摩尔2,4,6-TCP消耗2摩尔NADH和1摩尔双氧，反应产物鉴定为2,6-二氯对苯二酚。测定了辅因子和多种底物的稳态动力学参数。在另外两种底物饱和的条件下，FAD、NADH和2,6-二氯苯酚（2,6-DCP）的低K(m)值分别为1±0.1 microM、32±5 microM和4±2 microM。在以2,6-DCP为底物时，甲巯咪唑（MMI）对该酶有竞争性抑制作用，K(i)=27 microM。当研究其他多氯底物时，发现MMI的IC(50)值与其表观亲和力范围相符。基于芳香产物的形成，讨论了2,4,6-TCP和2,4,5-TCP降解的NADH和O(2)消耗方案。Blast搜索显示，这种酶与皮氏伯克霍尔德菌和固氮菌属GP1菌株的2,4,6-TCP-4-单加氧酶具有较高的序列同一性（60%），它们都催化对位羟基化脱卤反应。
