一种二苯醚类污染物直接开环角度双加氧酶的分子机制研究

Angular dioxygenation is the key degradation step of many toxic recalcitrant aromatic ring compounds containing the angular carbon atom such as dioxin, diphenyl ether, dibenzofuran and carbazole. The applicant acquired a novel direct cleavage angular dioxygenase Dpe which catalyze the angular dioxygenation of diphenyl ether and 2-carboxy diphenyl ether, resulting in the ortho cleavage of the C-C bond between the angular carbon atom and the neighbor carbon atom, following the opening of one aromatic ring to form 2,4-hexadienal phenyl ester. This is quite different from the early reported angular dioxygenase catalyzing the meta cleavage of the C-O/N bond.The target of this project is to further identify the structure of the product of diphenyl ether and 2-carboxy diphenyl ether catalyzed by Dpe usingy nuclear magnetic resonance and isotope labeling method, study the organization and transcription of the dpe gene cluster, the enzyme characteristics, substrate spectrum and kinetics of Dpe, simulate the structure of Dpe and identify its activate site. This project will reveal the characteristics and function of a new angular dioxygenase, provide theoretical basis for intensive study of the molecular mechanism of diphenyl ether pollutants biodegradation and the microbial remediation of diphenyl ether pollutants.

角度双加氧是细菌对含角度碳原子的有毒难降解芳烃污染物如二噁英、二苯醚、二苯呋喃和咔唑等的关键降解步骤。申请人前期研究获得了一个新的直接开环角度双加氧酶并将其命名为Dpe（diphenyl ether，二苯醚），它能够催化二苯醚角度C原子和邻位C原子之间C-C键断裂，芳环直接开环生成2,4-己二烯酸苯酚酯。这和已报道的催化间位C-O/N键自发断裂的角度双加氧酶不同。本项目拟利用核磁共振和同位素标记技术进一步鉴定双加氧断裂产物结构，研究dpe基因簇的组成和转录调控，Dpe酶学特性、底物谱和降解动力学，模拟Dpe酶的空间结构并筛选关键活性位点。本项目旨在为深入研究含角度碳芳烃类污染物的生物降解分子机制及生物修复技术研发提供理论依据。

含角度碳芳烃化合物广泛应用于农药、医药和化工原料及中间体的生产过程中。角度双加氧是细菌对含角度碳原子的有毒难降解芳烃污染物如二噁英、二苯醚、二本并呋喃和咔唑等的关键降解步骤。已报道的由细菌内酶催化的该角度双加氧反应导致角度碳原子和中间环上的杂原子之间的化学键断裂（间位断裂），在这一过程中芳环不断裂。Dpe是申请人前期研究获得的一个新的直接开环角度双加氧酶，它能够催化二苯醚角度碳原子和邻位碳原子之间的C-C键断裂，芳环直接开环生成2，4-己二烯酸苯酚酯。这和已报道的催化间位C-O/N键自发断裂的角度双加氧酶不同。本项目以Dpe酶为研究对象，通过底物利用和核磁共振技术进一步鉴定了双加氧断裂产物结构；通过比较基因组学及同源重组敲除手段推测dpe基因簇各个ORF的功能；研究该酶的生物学特性及降解动力学，通过空间模拟筛选其关键活性位点。本项目从生化和分子生物学角度揭示一种新的直接开环角度双加氧的反应机制，为深入研究含角度碳芳烃类污染物的生物降解分子机制及生物修复技术研发提供理论依据。