The goals of the proposed work are to obtain data that will allow descriptions at the molecular level of how the terminal two heme synthetic pathway enzymes, protoporphyrinogen oxidase (PPO) and ferrochelatase, function in both eucaryotes and procaryotes. We believe that this is an important research area for which there exist significant information gaps. Recently the importance of cellular heme homeostasis has garnered additional interest with the identification of a number of central metabolic pathways for which heme is a recognized cofactor or regulatory ligand. In addition, dysfunction at the level of the terminal two steps can result not only in diminished heme synthesis which may compromise regulatory networks, but also in the accumulation of the potentially toxic substrates (ferrous iron and protoporphyrin) and products (heme) of the reactions. The data acquired from the proposed work will also identify and characterize key differences that exist between the human and microbial enzymes which may, in the future, set the stage for development of new classes of antimicrobial agents.
Specific aims of the current proposal are to: 1) extend our structure/function studies on the terminal heme biosynthetic enzymes, 2) examine the potential role of the [2Fe-2S] cluster in ferrochelatase, 3) define protein- protein interactions involving ferrochelatase,4) characterize the oxygen independent protoporphyrinogen oxidases in Gram - bacteria, 5) identify the 'missing' bacterial coproporphyrinogen oxidases and 3) characterize essential heme biosynthetic protein(s) unique to the high G/C Gram + bacteria, Actinobacteria. Experimental approaches to be employed include classical and modern protein structure-function and protein-protein interaction techniques. Some of the proposed x-ray structure determination, biophysical characterizations and zebrafish-based work will be done via arranged collaborations, but the majority of work will be carried out in the PI's lab.
Given the number of regulatory networks modulated by heme, it is not surprising that in individuals with significant impairment in the ability to synthesize heme one finds developmental abnormalities. The proposed work will add significantly to the basic knowledge of heme synthesis and, thereby, address issues such as dysfunctional circadian rhythm (which has been suggested to be associated with psychiatric disorders and cancer) metabolic syndrome, atherosclerosis and neuronal decay of aging.
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