Biochemical, genetics and molecular biology of the bacterial biphenyl catabolic pathway enzymes

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Chirality is a key factor in the efficacy of many drugs and agrochemicals. In addition, complex compounds such as hydroxylated heterocyclic aromatics and flavonoids are regarded as very promising for the prevention and treatment of cancers and cardio-vascular diseases. Plants are currently the major source for these chemicals, but the synthesis of novel derivatives exhibiting improved biological properties is often difficult or impractical. Furthermore, in the context of the green chemistry concept, new more selective and more environmentally friendly approaches to manufacture these biologically specific fine chemicals will be required. This includes the use of biocatalysts to catalyze stereospecific reactions. In the present proposal we will focus on the biochemical properties of the biphenyl dioxygenase (BPDO), which catalyses the stereospecific dioxygenation of biphenyl to generate a cis-dihydrodiol metabolite. The substrates for BPDOs include many benzene or diphenyl skeletons, whose hydrogens are substituted with either methyl, ethyl, vinyl, carboxyl, halogenated or nitro groups. It can also oxygenate to cis-diol bicyclic- or tricyclic-fused heterocyclic aromatics such as quinoline, dibenzofuran and phenanthridine. Understanding how the BPDO catalytic pocket interacts with the substrate (and substrate analogs) to bind them and orient them into the catalytic pocket and understanding the mechanisms by which the enzyme can evolve to enhance its specificity toward new substrate analogs will help design novel biocatalysts useful in biotechnological processes for the destruction of persistent pollutants or biocatalytic processes for green production of chemicals. The objectives of this proposal are to pursue with some of the basic investigations:  A- regarding the biochemistry of the BPDO to get a better insight about the mechanism of catalytic activity and B- regarding the dynamics of BPDO evolution, answering specific questions about the protein domains and amino acid residues representing the major determinants of substrate specificity, regiospecificity and stereospecificity and how they interact with the substrate.