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功效的关键因素。此外，复杂化合物如羟基化杂环芳香族化合物和黄酮类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品的主要来源，但合成具有改善的生物学特性的新型衍生物通常是困难或不切实际的。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境友好的方法来制造这些生物特异性精细化学品。这包括使用生物催化剂来催化立体特异性反应。在本研究中，我们将重点研究联苯双加氧酶（BPDO）的生物化学性质，它催化联苯的立体定向双加氧反应生成顺式二氢二醇代谢产物。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。它也可以是顺式-顺式-二醇双环-或三环-稠合的杂环芳族化合物，如喹啉、二苯并呋喃和菲啶。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，并了解酶可以进化以增强其对新底物类似物的特异性的机制，将有助于设计用于破坏持久性污染物的生物技术过程或用于化学品的绿色生产的生物催化过程的新型生物催化剂。本建议的目标是进行一些基本调查：A-关于BPDO的生物化学，以更好地了解催化活性的机制，以及B-关于BPDO进化的动力学，回答关于代表底物特异性的主要决定因素的蛋白质结构域和氨基酸残基的具体问题，区域特异性和立体特异性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.