Research On The Structure And Function Of Caenorhabditis Elegans Mitochondrial Very Long Chain Acy1-CoA Dehydrogenase, Enoy1-CoA Hydratase And Thiolase

The mitochondrial P-oxidation spiral of fatty acid degradation consists of four enzymatic reactions that reduce the acyl chain length by two carbons in each cycle. The four enzymes that drive this cycle in mitochondria are acyl-CoA dehydrogenases(ACADs), enoyl-CoA hydratases(ECHs), (L)-3-hydroxyacyl-CoA dehydrogenase, and thiolases(THLs). In the initial step of the mitochondrialβ-oxidation spiral, acyl-CoA dehydrogenases catalyze the a, P-dehydrogenation of acyl-CoAs with the formation of the trans-2,3-enoyl-CoA products. Enoyl-CoA hydratases catalyze the reversible syn-hydration of trans-2,3-enoyl-CoA thioesters to the corresponding (L)-3-hydroxyacyl-CoA thioesters. Then in the third step (L)-3-hydroxyacyl-CoA dehydrogenases catalyze the oxidation of the hydroxyl group of (L)-hydroxyacyl-CoA to a keto group resulting in the formation of 3-ketoacyl-CoA thioesters. Finally, thiolases catalyze the reversible cleavage reaction of 3-ketoacyl-CoA to yield acetyl-CoA and an acyl-CoA molecule that has been shortened by two carbon atoms.There are many metabolic diseases related to the deficiency of the mitochondrial fatty acid P-oxidation, such as metabolic acidosis, hyperammonemia, fatty liver and diabetes mellitus, etc. Recently, there has been much and much concerns on the research of the mechanism and control of P-oxidation. But there is few report about the structure and function of the mitochondrialβ-oxidation of Caenorhabditis elegans which could be a good model organism for researching human diseases. Here three kinds of genes that are related to the fatty acid metabolism of Caenorhabditis elegans were cloned and overexpressed in Escherichia coli strain BL21(DE3). These genes are Y45F3A.3, C32E8.9 and F53A2.7 that express Caenorhabditis elegans mitochondrial very long chain acyl-CoA dehydrogenase(cVLCAD), enoyl-CoA hydratase(cECH) and thiolase(cTHL) respectively, according to the message of WormBase. The target proteins were purified to high purity and the crystals with high quality were obtained after exhaustive crystallization. And the high-resolution diffraction data sets were collected on the beamline BL17A and beamline 5A, respectively.The crystal structure of cVLCAD complexed with C11-CoA was determined to 1.9 A resolution. The novel tetrameric polymerization of cVLCAD that have not been found in other very long chain acyl-CoA dehydrogenase yet were confirmed by SEC experiment and its tetrameric structure. In vitro assay for cVLCAD function indicated that its tetrameric structure was indeed its active state for dehydrogenation catalyzed by cVLCAD. The structure of cVLCAD complexed with the substrate C11-CoA and the function assay indicated that cVLCAD would change the conformation of its substrate-binding site to enlarge the vacuity for binding the acyl-CoAs with the carbon length longer than 12.The cECH crystal belonged to space group P21 with unit-cell parameters a= 138.6, b=116.7, c=115.3(?),α=β=90.0,γ=124.0°. The analytic results of its diffraction data set indicated that the cECH crystal was not a normal twin crystal but a "tri-crystal" made up of three sets of lattices. On the basis of this hypothesis, we determined the crystal structure of cECH to 2.1 (?) resolution by compiling a "detri" program according to the principle of detwinning. SEC experiment indicated that cECH is a trimer and different from the polymerization of other ECHs.The crystal structure of cTHL and cTHL complexed with CoA were determined to 2.5 A and 2.56 A, respectively. cTHL is a homotetramer with a subunit～43-kDa and structural analysis indicated that cTHL is a biosynthetic thiolase. A series of function assay for cTHL wild type and its mutants indicated that the tetrameric structure of cTHL is necessary for the cleavage reaction of acetoacetyl-CoA. The structure analysis indicated that cTHL tetrameric structure was stablized by the hydrophobic interaction between the 129-143 loops and structural analysis indicated that the deletion of the 129-143 loop could enlarge the substrate-binding pocket of cTHL and could transform the function of cTHL from synthetic thiolase to degradative thiolase.