Wieser M, Wagner B, Eberspächer J, Lingens F
Institut für Mikrobiologie, Universität Hohenheim, Stuttgart, Germany.
J Bacteriol. 1997 Jan;179(1):202-8. doi: 10.1128/jb.179.1.202-208.1997.
The enzyme which catalyzes the dehalogenation of 2,4,6-trichlorophenol (TCP) was purified to apparent homogeneity from an extract of TCP-induced cells of Azotobacter sp. strain GP1. The initial step of TCP degradation in this bacterium is inducible by TCP; no activity was found in succinate-grown cells or in phenol-induced cells. NADH, flavin adenine dinucleotide, and O2 are required as cofactors. As reaction products, 2,6-dichlorohydroquinone and Cl- ions were identified. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of O2 per mol of TCP and the formation of 1 mol of Cl- ions. No evidence for membrane association or for a multicomponent system was obtained. Molecular masses of 240 kDa for the native enzyme and 60 kDa for the subunit were determined, indicating a homotetrameric structure. Cross-linking studies with dimethylsuberimidate were consistent with this finding. TCP was the best substrate for 2,4,6-trichlorophenol-4-monooxygenase (TCP-4-monooxygenase). The majority of other chlorophenols converted by the enzyme bear a chloro substituent in the 4-position. 2,6-Dichlorophenol, also accepted as a substrate, was hydroxylated in the 4-position to 2,6-dichlorohydroquinone in a nondehalogenating reaction. NADH and O2 were consumed by the pure enzyme also in the absence of TCP with simultaneous production of H2O2. The NH2-terminal amino acid sequence of TCP-4-monooxygenase from Azotobacter sp. strain GP1 revealed complete identity with the nucleotide-derived sequence from the analogous enzyme from Pseudomonas pickettii and a high degree of homology with two nondehalogenating monooxygenases. The similarity in enzyme properties and the possible evolutionary relatedness of dehalogenating and nondehalogenating monooxygenases are discussed.
从固氮菌属菌株GP1的2,4,6-三氯苯酚（TCP）诱导细胞提取物中纯化出催化TCP脱卤的酶，达到了表观均一性。该细菌中TCP降解的起始步骤可被TCP诱导；在琥珀酸生长的细胞或苯酚诱导的细胞中未发现活性。需要NADH、黄素腺嘌呤二核苷酸和O2作为辅因子。作为反应产物，鉴定出了2,6-二氯对苯二酚和Cl-离子。化学计量学研究表明，每摩尔TCP消耗2摩尔NADH加1摩尔O2，并形成1摩尔Cl-离子。未获得膜结合或多组分系统的证据。测定了天然酶的分子量为240 kDa，亚基分子量为60 kDa，表明其为同四聚体结构。用亚胺基二甲酯进行的交联研究与这一发现一致。TCP是2,4,6-三氯苯酚-4-单加氧酶（TCP-4-单加氧酶）的最佳底物。该酶转化的大多数其他氯酚在4位带有氯取代基。同样被接受为底物的2,6-二氯苯酚在4位羟基化为2,6-二氯对苯二酚，这是一个非脱卤反应。在没有TCP的情况下，纯酶也会消耗NADH和O2，同时产生H2O2。固氮菌属菌株GP1的TCP-4-单加氧酶的NH2末端氨基酸序列与皮氏假单胞菌类似酶的核苷酸衍生序列完全相同，与两种非脱卤单加氧酶具有高度同源性。讨论了脱卤和非脱卤单加氧酶在酶性质上的相似性以及可能的进化相关性。
