Remodeling Rieske-type bacterial dioxygenases to change catalytic properties

重塑 Rieske 型细菌双加氧酶以改变催化特性

• 批准号: 39579-2012
• 负责人: Sylvestre, Michel
• 金额: $ 1.89万
• 依托单位: Institut national de la recherche scientifique
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=568827
• 关键词: Remodeling; Rieske; type; bacterial; dioxygenases

The degradation of aromatic compounds by bacteria is initiated by a dioxygenation reaction catalyzed by an enzyme belonging to the Rieske-type aryl hydroxylating dioxygenases (ROs) to generate a cis-diol metabolite. The biphenyl dioxygenase (BPDO) that catalyzes the first step of the biphenyl catabolic pathway provides an example of RO. ROs exhibit broad substrates ranges. Besides their potential contribution to the bioremediation technologies, the regio- and stereo-chemical reactions catalyzed by ROs are of special interest because of the growing demand for single isomers of chiral pharmaceuticals. One special group of chemicals of great interest are the flavonoids derived from plants. Our recent investigations have principally involved Pandoraea pnomenusa B-356 and Burkholderia xenovorans LB400 BPDOs and variants derived from them. Using semi-rational approaches of directed evolution we have engineered novel BPDOs that oxygenate a broad range of BPH analogs including polychlorinated biphenyls (PCBs) and chlorodibenzofurans more efficiently than the parental enzymes. We have compared the biochemical and structural features of these enzymes to identify some of the mechanisms by which they evolve to expand their substrate range. The objectives of the current proposal are to identify the protein domains and amino acid residues representing the major determinants of substrate specificity, regiospecificity and stereospecificity and to determine how they interact together to shape the catalytic pocket and how they interact with the substrate to position it inside the catalytic pocket. This will provide better insights about the bases for BPDO broad substrate range and about the mechanisms by which the enzyme evolve to expand its substrate range and its regiospecificity. We will focus our investigation on the interaction between BPDO and PCBs and flavonoids. From a practical standpoint this investigation will help in designing strategies to engineer new enzymes exhibiting enhanced activities toward BPH analogs. This proposal is to be placed in the context of the growing importance of biocatalytic processes for the restoration of contaminated sites and for the green-chemistry-based processes to synthesize new more specific drugs.

细菌对芳香化合物的降解是由属于Rieske型芳基芳基羟基二加氧酶（ROS）催化的二氧化反应引发的，从而产生CIS-二醇代谢物。催化双苯基分解代谢途径的第一步的双苯基二氧酶（BPDO）提供了RO的示例。 ROS表现出广泛的底物范围。除了对生物修复技术的潜在贡献外，由于对手性药物的单个异构体的需求不断增长，因此特别感兴趣的是由ROS催化的区域和立体化学反应。一个特别感兴趣的化学品是源自植物的类黄酮。我们最近的调查主要涉及Pandoraea Pnomenusa B-356和Burkholderia Xenovorans LB400 BPDOS和从中得出的变体。使用定向进化的半理性方法，我们对新型的BPDO进行了设计，这些新型bpdos比亲属酶更有效地更有效地氧化了包括多氯联苯和氯二苯呋喃的广泛的BPH类似物。我们已经比较了这些酶的生化和结构特征，以确定它们发展以扩大其底物范围的某些机制。当前建议的目标是确定代表蛋白质结构域和氨基酸残基，这些蛋白质域和氨基酸残基代表了底物特异性，重型特异性和立体特异性的主要决定因素，并确定它们如何共同相互作用以形成催化口袋以及如何与底物相互作用以将其与底物相互作用以将其放置在催化口袋内。这将提供有关BPDO广泛底物范围基础的更好的见解，以及酶扩大其底物范围及其恢复特定性的机制。我们将把调查集中在BPDO与PCB和类黄酮之间的相互作用上。从实际的角度来看，此调查将有助于设计策略，以设计新的酶，展现出对BPH类似物的增强活动。该提案应置于生物催化过程对恢复受污染部位的重要性以及基于绿色化学的过程以综合新的更特定药物的情况下的重要性。