Remodeling Rieske-type bacterial dioxygenases to change catalytic properties

细菌对芳香族化合物的降解是由rieske型芳基羟基化双加氧酶（ROs）催化的双加氧反应引发的，产生顺式二醇代谢物。催化联苯分解代谢途径第一步的联苯双加氧酶（BPDO）是反渗透的一个例子。活性氧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，由于对手性药物单异构体的需求日益增长，由活性氧催化的区域和立体化学反应受到特别关注。从植物中提取的类黄酮是一类特别有趣的化学物质。我们最近的研究主要涉及现象潘多拉菌B-356和异种伯克霍尔德菌LB400 bpdo及其衍生变体。利用定向进化的半理性方法，我们设计了新型双酚类化合物，它比亲本酶更有效地氧化广泛的双酚类化合物类似物，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。当前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袋，以及它们如何与底物相互作用以将其定位在催化袋内。这将为BPDO广泛底物范围的基础以及酶进化以扩大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与多氯联苯和类黄酮的相互作用。从实际的角度来看，这项研究将有助于设计策略来设计对BPH类似物具有增强活性的新酶。这一建议是在生物催化过程对污染场地的恢复和基于绿色化学的过程合成新的更具体的药物日益重要的背景下提出的。

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.