Biochemistry of Two FADH2-Utilizing Trichlorophenol 4-Monooxygenases

多氯酚是一组主要的污染物，在上个世纪，多氯酚通过在消费品、木材工业和农业中的应用而被引入到环境中。由于具有特定取代基的化合物的特殊用途，以及由于某些制造工艺缺乏特异性，因此存在许多多氯苯酚异构体。通常，卤化物取代的重要性在于，它通过使化合物相对不易于微生物降解来增加化合物的持久性。在制造持久性化合物方面取得的太多成功导致其在环境中积累。目前正在努力鉴定和开发能够降解一些问题较多的多氯酚的微生物。PI小组的研究为全面阐明三种重要的多氯苯酚的降解途径做出了重大贡献：2，4，6-三氯苯酚、2，4，5-三氯苯酚和五氯苯酚。已经发现了一组新的利用FADH 2的氧化多氯酚的单加氧酶。两种密切相关的利用FADH 2的单加氧酶TcpA和TftD分别启动2，4，6-TCP和2，4，5-TCP的降解。最近的数据表明，TcpA催化氧化和水解反应的两个脱氯步骤，而TftD顺序脱氯2，4，5-TCP的两个氧化反应。第一个研究目标是证明拟议的脱氯机制。第二个目标是本地化的氨基酸残基负责改变催化的高度同源的TftD和TcpA的定制结构域交换和定点诱变策略。这些结果将导致对酶通过突变和选择来降解新化合物的方式进行更完整的描述。该研究旨在为利用FADH 2的单加氧酶在环境污染物生物修复中的应用提供科学指导。 该项目研究了微生物如何进化以降解多氯酚的各种异构体，这是一组高毒性污染物。该项目还研究了微生物降解这些化合物的反应机制。该研究成果将为多氯酚污染物的治理提供科学指导，具有广泛的社会影响。这项研究还将对生物化学、微生物学和进化理论产生直接影响。通过这项研究，一名研究生将接受培训，在生物化学和微生物学进行独立的研究。一些本科生将在项目的某些方面工作，以获得研究经验。这些教育工作将使学生做好准备，迎接生物和环境科学的新挑战。

Polychlorophenols are a major group of pollutants, which have been introduced into the environment over the last century through their applications in consumer products, wood industry, and agriculture. Due to special uses for compounds with particular substitutions and due to lack of specificity in some of the manufacturing processes, many polychlorophenol isomers exist. Often the importance of a halide substitution is that it increases the persistence of a compound by making it relatively recalcitrant to microbial degradation. Too much success in making a persistent compound has led to its accumulation in the environment. Efforts are underway to identify and develop microorganisms that can degrade some of the more problematic polychlorophenols. Research from the PI's group has significantly contributed to the complete elucidation of degradation pathways for three important polychlorophenols: 2,4,6-trichlorophenol (2,4,6-TCP), 2,4,5-trichlorophenol (2,4,5-TCP), and pentachlorophenol. A novel group of FADH2-utilizing monooxygenases that oxidize polychlorophenols has been discovered. Two closely related FADH2-utilizing monooxygenases, TcpA and TftD, initiate the degradation of 2,4,6-TCP and 2,4,5-TCP, respectively. Recent data suggests that TcpA catalyzes both oxidative and hydrolytic reactions for two dechlorination steps, whereas TftD sequentially dechlorinates 2,4,5-TCP by two oxidation reactions. The first research objective is to demonstrate the proposed dechlorination mechanisms. The second objective is to localize the amino acid residues responsible for the altered catalysis of the highly homologous TftD and TcpA by a tailored domain swapping and site-directed mutagenesis strategy. The results would lead to a more complete description of the way, in which enzymes are recruited by mutation and selection to degrade novel compounds. The proposed research aims to provide scientific guidance for applying FADH2-utilizing monooxygenases in bioremediation of environmental pollutants. The project investigates how microorganisms are evolving to degrade various isomers of polychlorophenols, a group of highly toxic pollutants. The project also studies the reaction mechanisms employed by microorganisms to degrade these compounds. The research will have a broad impact on society because the results will provide scientific guidance for the remediation of polychlorophenols. The research will also have a direct impact on biochemistry, microbiology, and evolution theories. Through this research, a graduate student will be trained to perform independent research in biochemistry and microbiology. Several undergraduate students will work on some aspects of the project to gain research experience. These educational efforts will prepare students to meet new challenges in biological and environmental sciences.