Research Project
2193785
Heme is the prosthetic group of cytochromes, hemoglobin, myoglobin and other respiratory enzymes, and precursors of the heme biosynthetic pathway are also used for the synthesis of chlorophylls, vitamin B12 and bilins in organisms that express them. Defects in the genes of the heme metabolic pathway result in the inability of bacteria to grow in minimal media and in the human results in the diseases collectively known as porphyria. Heme biosynthesis from the first universally committed precursor, d- aminolevulinic acid (ALA) requires seven enzymatic steps. The genes encoding the three enzymes that convert ALA to Uroporphyrinogen III (Uro III, the first closed-ring tetrapyrrole intermediate in this universal pathway) have been isolated and characterized in bacterial, yeast and mammalian systems. The genes for three enzymes, glutamyl tRNA reductase, porphobilinogen deaminase and Uro III cosynthase from the green sulfur bacterium Chlorobium have been cloned and characterized. Uro III is converted to protoheme IX in four enzymatic steps, catalyzed by Uro III decarboxylase, coproporphyrinogen III oxidase, protoporphyrinogen IX oxidase and ferrochelatase (encoded by hemE, F, G and H, respectively). Compared to the pre-Uro III genes, much less genetic information is available about these four enzymes and the corresponding genes. There is no published sequence data on hemG from any organism and the hemF gene from only a few organisms has been cloned and sequenced. The cloning and expression studies of the hemE, F, G and H genes from Chlorobium are being proposed in this application. The following experiments will be performed: a) construction of a genomic library, b) identification o of the specific cloned genes by genetic complementation of corresponding E. coli and/or B. subtilis heme auxotrophs, c) growth analysis of the complemented auxotrophs followed by respective enzyme assays, and d) Southern analysis and sequencing. These will be followed by Northern analysis and coupled in vitro transcription-translation for RNA characterization and peptide analysis, respectively. These studies will provide substantial information on the structure and expression of the hem genes. While there are obstacles and handling difficulties in using the human system the established Chlorobium system is ideal for students to learn about this essential pathway and will provide with general information that may be applicable to human.
