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Reconstitution, morphology and crystallization of a fatty acid beta-oxidation multienzyme complex from Pseudomonas fragi.

ABSTRACT: The fatty acid beta-oxidation multienzyme complex from Pseudomonas fragi, HDT, exhibits predominantly the three enzymic activities of 2-enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The HDT complex is encoded by the faoAB operon, consisting of the faoA and faoB genes that encode two individual constituents, the alpha-subunit and the beta-subunit. We have constructed Escherichia coli overexpression systems for the faoAB gene product (coexpression of the alpha- and beta-subunits), the alpha-subunit alone and the beta-subunit alone, and have purified the three respective products. Gel-filtration analysis revealed that the faoAB gene product forms a heterotetrameric structure, alpha2beta2, identical with the native HDT oligomeric state from P. fragi, whereas the alpha-subunit and beta-subunit individually form dimers. Electron microscopy demonstrated that each protein morphologically adopts the above oligomeric structures. The HDT complex, reconstituted in vitro from the isolated alpha- and beta-subunits, exhibits the three original enzymic activities and yields the same crystal as those from the native enzyme. CD measurements indicated that the alpha- and beta-dimers hardly alter their global conformations upon the formation of the HDT complex. Interestingly, the beta-dimer alone does not exhibit 3-oxoacyl-CoA thiolase activity, whereas the alpha-dimer alone exhibits both the 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities. These results suggest that the contact between the alpha- and beta-subunits is essential for the thiolase activity. We have identified several structurally important proteolytic sites within each subunit, which are protected in the intact heterotetrameric molecule. These findings allow the possible location of the interface between the two subunits, which should be crucial for the exhibition of thiolase activity.

SUBMITTER: Ishikawa M

PROVIDER: S-EPMC1218991 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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Project description:Mutations in nuclear genes encoding for mitochondrial proteins very long-chain acyl-CoA dehydrogenase (VLCAD) and trifunctional protein (TFP) cause rare autosomal recessive disorders. Studies in fibroblasts derived from patients with mutations in VLCAD and TFP exhibit mitochondrial defects. To gain insights on pathological changes that account for the mitochondrial deficits we performed quantitative proteomic, biochemical, and morphometric analyses in fibroblasts derived from subjects with three different VLCAD and three different TFP mutations. Proteomic data that was corroborated by antibody-based detection, indicated reduced levels of VLCAD and TFP protein in cells with VLCAD and TFP mutations respectively, which in part accounted for the diminished fatty acid oxidation capacity. Decreased mitochondrial respiratory capacity in cells with VLCAD and TFP mutations was quantified after glucose removal and cells with TFP mutations had lower levels of glycogen. Despite these energetic deficiencies, the cells with VLCAD and TFP mutations did not exhibit changes in mitochondria morphology, distribution, fusion and fission, quantified by either confocal or transmission electron microscopy and corroborated by proteomic and antibody-based protein analysis. Fibroblasts with VLCAD and to a lesser extend cells with TFP mutations had increased levels of mitochondrial respiratory chain proteins and proteins that facilitate the assembly of respiratory complexes. With the exception of reduced levels of catalase and glutathione S-transferase theta-1 in cells with TFP mutations, the levels of 45 proteins across all major intracellular antioxidant networks were similar between cells with VLCAD and TFP mutations and non-disease controls. Collectively the data indicate that despite the metabolic deficits, cells with VLCAD and TFP mutations maintain their proteomic integrity to preserve cellular and mitochondria architecture, support energy production and protect against oxidative stress.

Project description:Mutations in nuclear genes encoding for mitochondrial proteins very long-chain acyl-CoA dehydrogenase (VLCAD) and trifunctional protein (TFP) cause rare autosomal recessive disorders. Studies in fibroblasts derived from patients with mutations in VLCAD and TFP exhibit mitochondrial defects. To gain insights on pathological changes that account for the mitochondria l deficits we performed quantitative proteomic, biochemical, and morphometric analyses in fibroblasts derived from subjects with three different VLCAD and three different TFP mutations. Proteomic data that was corroborated by antibody-based detection, indicated reduced levels of VLCAD and TFP protein in cells with VLCAD and TFP mutations respectively, which in part accounted for the diminished fatty acid oxidation capacity. Decreased mitochondrial respiratory capacity in cells with VLCAD and TFP mutations was quantified after glucose removal and cells with TFP mutations had lower levels of glycogen. Despite these energetic deficiencies, the cells with VLCAD and TFP mutations did not exhibit changes in mitochondria morphology, distribution, fusion and fission, quantified by either confocal or transmission electron microscopy and corroborated by proteomic and antibody-based protein analysis. Fibroblasts with VLCAD and to a lesser extend cells with TFP mutations had increased levels of mitochondrial respiratory chain proteins and proteins that facilitate the assembly of respiratory complexes. With the exception of reduced levels of catalase and glutathione S-transferase theta-1 in cells with TFP mutations, the levels of 45 proteins across all major intracellular antioxidant networks were similar between cells with VLCAD and TFP mutations and non-disease controls. Collectively the data indicate that despite the metabolic deficits, cells with VLCAD and TFP mutations maintain their proteomic integrity to preserve cellular and mitochondria architecture, support energy production and protect against oxidative stress.

Project description:BackgroundThe oxidation of fatty acids in mitochondria plays an important role in energy metabolism and genetic disorders of this pathway may cause metabolic diseases. Enzyme deficiencies can block the metabolism at defined reactions in the mitochondrion and lead to accumulation of specific substrates causing severe clinical manifestations. Ten of the disorders directly affecting mitochondrial fatty acid oxidation have been well-defined, implicating episodic hypoketotic hypoglycemia provoked by catabolic stress, multiple organ failure, muscle weakness, or hypertrophic cardiomyopathy. Additionally, syndromes of severe maternal illness (HELLP syndrome and AFLP) have been associated with pregnancies carrying a fetus affected by fatty acid oxidation deficiencies. However, little is known about fatty acids kinetics, especially during fasting or exercise when the demand for fatty acid oxidation is increased (catabolic stress).ResultsA computational kinetic network of 64 reactions with 91 compounds and 301 parameters was constructed to study dynamic properties of mitochondrial fatty acid beta-oxidation. Various deficiencies of acyl-CoA dehydrogenase were simulated and verified with measured concentrations of indicative metabolites of screened newborns in Middle Europe and South Australia. The simulated accumulation of specific acyl-CoAs according to the investigated enzyme deficiencies are in agreement with experimental data and findings in literature. Investigation of the dynamic properties of the fatty acid beta-oxidation reveals that the formation of acetyl-CoA - substrate for energy production - is highly impaired within the first hours of fasting corresponding to the rapid progress to coma within 1-2 hours. LCAD deficiency exhibits the highest accumulation of fatty acids along with marked increase of these substrates during catabolic stress and the lowest production rate of acetyl-CoA. These findings might confirm gestational loss to be the explanation that no human cases of LCAD deficiency have been described.ConclusionIn summary, this work provides a detailed kinetic model of mitochondrial metabolism with specific focus on fatty acid beta-oxidation to simulate and predict the dynamic response of that metabolic network in the context of human disease. Our findings offer insight into the disease process (e.g. rapid progress to coma) and might confirm new explanations (no human cases of LCAD deficiency), which can hardly be obtained from experimental data alone.

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Reconstitution, morphology and crystallization of a fatty acid beta-oxidation multienzyme complex from Pseudomonas fragi.

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