GENETIC ANALYSIS OF MG-TETRAPYRROLE BIOSYNTHESIS

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The complex tetrapyrrole biosynthetic pathway is responsible for synthesizing important metabolites such as vitamin B12, hemes, bilins and chlorophylls. The "common trunk" of the pathway from 5-aminolevulinate to protoporphyrin IX, has received the most attention owing to the fact that a number of heredity diseases (porphyrias) are caused by the overproduction of heme precursors. Clinical manifestations of overproducing these intermediates range from simple skin lesions, to psychotic disorders, to death. The vitamin B12 branch of the pathway has also received considerable attention as of late. Many genes involved in vitamin B12 synthesis have been identified in Pseudomonas dentrificans and in Salmonella typhimurium. In contrast to the information on heme and vitamin B12 synthesis, there is little information on genes which control synthesis of the Mg-tetrapyrrole branch which is responsible for synthesis of chlorophylls and bacteriochlorophylls. In this proposal we plan to perform detailed biochemical and genetic analysis of the Mg-tetrapyrrole biosynthetic pathway from the bacterium Rhodobacter capsulatus. This includes (i) Biochemical characterization of enzymes from the Mg-tetrapyrrole branch of the biosynthetic pathway, (ii) Biochemical and genetic characterization of a redox responding transcription factor that regulates expression of heme, Mg-tetrapyrrole and carotenoid biosynthesis genes as well as polypeptides that comprise the light harvesting-II portion of the photosystem. (iii). Characterization of additional transcriptional and post-transcriptional regulatory factors that controls the flow of intermediates through this branch of the tetrapyrrole biosynthetic pathway. (iv) Investigations on Mg-tetrapyrrole biosynthesis in the cyanobacterium Synechocystis PCC 6803 will also be initiated for use in a comparative analysis as well as a model system for Mg-tetrapyrrole biosynthesis in eukaryotes. A thorough understanding of the tetrapyrrole biosynthetic pathway has some far ranging practical implications beyond an academic philosophical interest. These include some obvious commercial applications, such as the design of herbicides that target enzymes in the Mg tetrapyrrole pathway, and the health implications of overproducing tetrapyrrole endproducts such as vitamin B12 and heme. It should also not be overlooked that tetrapyrrole driven photosynthesis is the primary route of capturing and supplying energy to living cells and, consequently, it is the most important source of energy in our technological world.