Construction of Environmental Biremediation Enzymes Whose Catalysis is Regulated by Light and Molecular Switches

细胞色素P450激活致癌活性和毒性的各种环境化学品，包括苯并芘，黄曲霉毒素，卤代乙烷。然而，细胞色素P450也参与各种环境化学物质的代谢和降解。因此，利用细胞色素P450的降解特性，有可能构建高效、实用的环境生物修复酶。一氧化氮（NO）是一种重要的生理气体，由一氧化氮合酶合成，其活性部位结构与细胞色素P450相同。另一方面，有许多传感酶和传感蛋白，电子的引入、光照、钙离子、CO、NO、分子氧和各种致癌物的结合引起蛋白质构象的变化。这些变化成为传递到催化结构域的信号，并切换催化的开/关。在本研究中，我们研究了钙传感器蛋白，神经元NO合成酶的电子传递过程和蛋白质-蛋白质相互作用。然后，我们构建了由NO合酶的钙传感器结构域和细胞色素P450的Have结合活性位点组成的嵌合酶。我们用一种新的分子开关酶检测了所产生的钙传感器嵌合酶的电子转移和催化效率。发现嵌合酶作为钙传感器酶起作用。此外，我们研究了一种新的氧化还原传感器酶，Ec DOS的结构和功能的关系。结果发现，Ec DOS也是CO和NO传感器酶，因为催化强烈抑制CO和NO。作为本项目研究的最后一步，我们表征潜在的血红素传感器酶，SOUL。总之，本研究为构建具有高效降解环境化学物质特性的新型传感酶提供了可能。

Cytochrome P450s activate carcinogenetic activities and toxicities of various environmental chemicals including benzpyrene, aflatoxin, haloethanes. However, cytochrome P450s are also involved in metabolism and degradation of various environmental chemicals. Therefore, it is possible that we can construct efficient and useful environmental bioremediation enzymes if the degradation properties of cytochrome P450s can be used. A physiologically important gas, nitric oxide (NO), is synthesized by nitric oxide synthase whose active site structure is the sameas those of cytochrome P450s. On the other hand, there are many sensor enzymes and sensor proteins, where electron introduction, light illumination, bindings of calcium ion, CO, NO, molecule oxygen and various carcinogens induced certain protein conformational changes. The changes became signals that transmit to catalytic domain and switch the catalysis on/off. In the present study, we examined electron transfer process and protein-protein interactions of a calcium sensor protein, neuronal NO synthase. Then, we constructed chimeric enzymes composed of the calcium sensor domain of NO synthase and the have-bound active site of cytochrome P450. We examined efficiencies of electron transfer and catalysis of the generated calcium sensor chimeric enzymes with the an of a novel molecular switching enzyme. It was found that the chimeric enzyme functions as a calcium sensor enzyme. Furthermore, we examined structure and function relationships of a new redox sensor enzyme, Ec DOS. It was found that Ec DOS is also CO and NO sensor enzyme, because the catalysis was strongly inhibited by CO and NO. As the last step of this project study, we characterized potential heme-sensor enzyme, SOUL. In summary, the study provided the possibility to construct novel sensor enzymes with efficient biodegradation properties of environmental chemicals.