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

手性是许多药物和农用化学品疗效的关键因素。此外，羟基杂环芳烃和黄酮类化合物等复杂化合物被认为在预防和治疗癌症和心血管疾病方面非常有前途。植物目前是这些化学物质的主要来源，但合成具有改善生物特性的新型衍生品往往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生产这些具有生物特性的精细化学品。这包括使用生物催化剂来催化立体专一性反应。在本提案中，我们将重点介绍联苯双加氧酶(BPDO)的生化性质，该酶催化联苯的立体特异性双加氧生成顺式二氢二醇代谢物。BPDO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、卤代或硝基取代。它还可以氧化成顺二醇、双环或三环稠合杂环芳香族化合物，如喹啉、二苯并呋喃和菲。了解BPDO催化口袋如何与底物(和底物类似物)相互作用以结合它们并将它们定向到催化口袋中，并了解酶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新型生物催化剂，这些生物催化剂可用于破坏持久性污染物的生物技术过程或用于绿色生产化学品的生物催化过程。这项建议的目标是进行一些基础研究：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的进化动力学，回答关于蛋白质结构域和氨基酸残基的具体问题，这些蛋白质结构域和氨基酸残基代表底物专一性、区域专一性和立体专一性的主要决定因素以及它们如何与底物相互作用。

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.