Biochemical, genetics and molecular biology of the bacterial biphenyl catabolic pathway enzymes

手性是影响许多药物和农用化学品药效的关键因素。此外，羟基化杂环芳烃和类黄酮等复杂化合物在预防和治疗癌症和心血管疾病方面被认为是非常有前途的。植物目前是这些化学物质的主要来源，但合成具有改进生物特性的新型衍生物通常是困难的或不切实际的。此外，在绿色化学概念的背景下，将需要新的更具选择性和更环保的方法来制造这些具有生物特异性的精细化学品。这包括使用生物催化剂来催化立体特异性反应。在本提案中，我们将重点关注联苯双加氧酶（BPDO）的生化特性，它催化联苯的立体特异性双加氧生成顺式二氢二醇代谢物。双pdo的底物包括许多苯或二苯基骨架，其氢被甲基、乙基、乙烯基、羧基、卤化基或硝基取代。它还可以氧化成顺式二醇双环或三环熔接的杂环芳烃，如喹啉、二苯并呋喃和菲咯啶。了解BPDO催化袋如何与底物（和底物类似物）相互作用，将它们结合并将它们定向到催化袋中，以及了解酶如何进化以增强其对新底物类似物的特异性的机制，将有助于设计出在生物技术过程中有用的新型生物催化剂，用于破坏持久性污染物或绿色生产化学品的生物催化过程。本提案的目标是继续进行一些基础研究：A-关于BPDO的生物化学，以更好地了解催化活性的机制；B-关于BPDO进化的动力学，回答关于代表底物特异性，区域特异性和立体特异性的主要决定因素的蛋白质结构域和氨基酸残基以及它们如何与底物相互作用的具体问题。

Chirality is a key factor in the efficacy of many drugs and agrochemicals. In addition, complex compounds such as hydroxylated heterocyclic aromatics and flavonoids are regarded as very promising for the prevention and treatment of cancers and cardio-vascular diseases. Plants are currently the major source for these chemicals, but the synthesis of novel derivatives exhibiting improved biological properties is often difficult or impractical. Furthermore, in the context of the green chemistry concept, new more selective and more environmentally friendly approaches to manufacture these biologically specific fine chemicals will be required. This includes the use of biocatalysts to catalyze stereospecific reactions. In the present proposal we will focus on the biochemical properties of the biphenyl dioxygenase (BPDO), which catalyses the stereospecific dioxygenation of biphenyl to generate a cis-dihydrodiol metabolite. The substrates for BPDOs include many benzene or diphenyl skeletons, whose hydrogens are substituted with either methyl, ethyl, vinyl, carboxyl, halogenated or nitro groups. It can also oxygenate to cis-diol bicyclic- or tricyclic-fused heterocyclic aromatics such as quinoline, dibenzofuran and phenanthridine. Understanding how the BPDO catalytic pocket interacts with the substrate (and substrate analogs) to bind them and orient them into the catalytic pocket and understanding the mechanisms by which the enzyme can evolve to enhance its specificity toward new substrate analogs will help design novel biocatalysts useful in biotechnological processes for the destruction of persistent pollutants or biocatalytic processes for green production of chemicals. The objectives of this proposal are to pursue with some of the basic investigations: A- regarding the biochemistry of the BPDO to get a better insight about the mechanism of catalytic activity and B- regarding the dynamics of BPDO evolution, answering specific questions about the protein domains and amino acid residues representing the major determinants of substrate specificity, regiospecificity and stereospecificity and how they interact with the substrate.