Remodeling Rieske-type bacterial dioxygenases to change catalytic properties

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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.