Remodeling Rieske-type bacterial dioxygenases to change catalytic properties

细菌对芳香族化合物的降解是由Rieske型芳基羟化双加氧酶(ROS)催化的双加氧反应生成顺式二醇代谢物而开始的。联苯双加氧酶(BPDO)催化联苯分解代谢途径的第一步，是RO的一个例子。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，由于手性药物对单一异构体的需求日益增长，ROS催化的区域化学反应和立体化学反应也引起了人们的特别关注。从植物中提取的黄酮类化合物是一类特别令人感兴趣的化学物质。我们最近的研究主要涉及Pandoraea pnomenusa B-356和Burkholderia exovorans LB400 BPDO及其变异体。使用定向进化的半理性方法，我们设计了新型的BPDO，它比亲本酶更有效地氧化包括多氯联苯(PCB)和氯二苯并呋喃在内的广泛的BPH类似物。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。本提案的目标是确定代表底物特异性、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，并确定它们如何相互作用以形成催化口袋，以及它们如何与底物相互作用以将其定位在催化口袋内。这将为更好地了解BPDO广泛底物范围的碱基，以及酶进化以扩大其底物范围和区域特异性的机制提供更好的见解。我们将重点研究BPDO与多氯联苯和黄酮类化合物的相互作用。从实践的角度来看，这项研究将有助于设计策略来设计新的酶，使其对BPH类似物表现出更强的活性。这项建议将放在生物催化过程对修复受污染场地和以绿色化学为基础的过程合成新的、更具体的药物日益重要的背景下提出。

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.