This project is designed to answer a number of questions and provide information important for developing a detailed understanding of the beta-oxidation of polyunsaturated fatty acids. The focus of this study will be on 2,4-dienoyl-CoA reductase and 3-hydroxyacyl-CoA epimerase which are key enzymes in the reductase-dependent and epimerase-dependent pathways of PUFA beta-oxidation, respectively. Biochemical and genetic approaches will be used to study the beta-oxidation systems of mammals and E. coli. An important aspect of this study is an assessment of the importance of 3-hydroxyacyl-CoA epimerase in the beta-oxidation of hydroxy fatty acids like D-5-hydrooxyeicosatetraenoic acid (5-HETE) in mammals and D-3-hydroxymyristic acid in E. coli. Also, the mechanism of the E. coli 3-hydroxyacyl-CoA epimerase will be elucidated as will be the location of delta3-cis-delta2-trans-enoyl-CoA isomerase on the multienzyme complex of fatty acid oxidation from E. coli. The identification of a human disorder due to a deficiency of 2,4- dienoyl-CoA reductase prompts further studies of this enzyme including (a) the development of a more sensitive assay to measure reductase activities in fibroblasts and lymphoblasts; (b) the purification of the peroxisomal reductase from rat liver; (c) the cloning, sequencing and overexpression of the E. coli reductase gene; (d) a stereochemical study of the reduction catalyzed by mammalian 2,4-dienoyl-CoA reductase and (e) the hormonal regulation of this enzyme and its effect on PUFA beta- oxidation. Finally, the beta-oxidation of cis and trans unsaturated fatty acids in mitochondria will be studied with the aim of detecting and explaining differences in their degradation. Altogether, this project will provide a more complete view of the beta-oxidation of polyunsaturated fatty acids and of some hydroxy fatty acids in normal and diseased organisms including humans. The above outlined project was approved and is funded from 9/91-8/95 by NIH Grant HL30847.
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