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: 2013
• 资助国家: 加拿大
• 起止时间: 2013-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=521521
• 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.

细菌降解芳香族化合物是由属于Rieske型芳基羟基化双加氧酶（RO）的酶催化的双加氧反应引发的，以产生顺式二醇代谢物。催化联苯分解代谢途径的第一步的联苯双加氧酶（BPDO）提供了RO的实例。RO表现出广泛的底物范围。除了它们对生物修复技术的潜在贡献之外，由于对手性药物的单一异构体的需求不断增长，由RO催化的区域和立体化学反应特别令人感兴趣。一组特别令人感兴趣的化学物质是从植物中提取的类黄酮。我们最近的研究主要涉及Pandoraea pnomenusa B-356和Burkholderia xenovorans LB 400 BPDO及其衍生变体。使用半理性的定向进化的方法，我们已经设计了新的BPDO，其比亲本酶更有效地抑制广泛的BPH类似物，包括多氯联苯（PCB）和氯二苯并呋喃。我们比较了这些酶的生化和结构特征，以确定它们进化以扩大底物范围的一些机制。目前的建议的目的是确定蛋白质结构域和氨基酸残基代表底物特异性，区域特异性和立体特异性的主要决定因素，并确定它们如何相互作用在一起，形成催化口袋，以及它们如何与底物相互作用，将其定位在催化口袋内。这将提供关于BPDO广泛底物范围的基础以及关于酶进化以扩展其底物范围和其区域特异性的机制的更好见解。我们将重点研究BPDO与多氯联苯和黄酮类化合物之间的相互作用。从实用的角度来看，这项调查将有助于设计策略，工程新的酶表现出对BPH类似物的增强活性。这一建议是在生物催化过程对恢复受污染场地和基于绿色化学的过程对合成新的更具体的药物日益重要的背景下提出的。