Gisi Michelle R, Xun Luying
School of Molecular Biosciences, Washington State University, Pullman 99164-4324, USA.
J Bacteriol. 2003 May;185(9):2786-92. doi: 10.1128/JB.185.9.2786-2792.2003.
Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source. Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD). Sequence analysis suggests that they are separate enzymes. The two proteins were separately produced in Escherichia coli, purified, and characterized. TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase. A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate. Kinetic and binding property analysis showed that FAD was a better substrate than FMN. TftD was a reduced FAD (FADH(2))-utilizing monooxygenase, and FADH(2) was supplied by TftC. It converted 2,4,5-trichlorophenol to 2,5-dichloro-p-quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro-p-quinol as the final product. TftD interacted with FADH(2) and retarded its rapid oxidation by O(2). A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates. The reclassification of the two enzymes further supports the new discovery of FADH(2)-utilizing enzymes, which have homologues in the domains Bacteria and Archaea.
洋葱伯克霍尔德菌AC1100利用环境污染物2,4,5-三氯苯氧乙酸作为唯一碳源和能源。氯酚4-单加氧酶是2,4,5-三氯苯氧乙酸降解过程中的关键酶,最初被鉴定为一种双组分酶(TftC和TftD)。序列分析表明它们是独立的酶。这两种蛋白质在大肠杆菌中分别表达、纯化并进行了特性鉴定。TftC是一种NADH:黄素腺嘌呤二核苷酸(FAD)氧化还原酶。C末端带有组氨酸标签的融合TftC利用NADH还原FAD或黄素单核苷酸(FMN),但不将NADPH或核黄素用作底物。动力学和结合特性分析表明,FAD是比FMN更好的底物。TftD是一种利用还原型FAD(FADH₂)的单加氧酶,FADH₂由TftC提供。它将2,4,5-三氯苯酚转化为2,5-二氯对苯二酚,然后再转化为5-氯羟基喹啉,但将2,4,6-三氯苯酚仅转化为2,6-二氯对苯二酚作为最终产物。TftD与FADH₂相互作用并抑制其被O₂快速氧化。鉴定出了一系列可能与TftD结合的FAD-过氧化物,表明过氧化物可能是攻击芳香族底物的活性氧物种。这两种酶的重新分类进一步支持了利用FADH₂的酶的新发现,这类酶在细菌和古菌结构域中存在同源物。