Expression, purification, and characterization of His-tagged human mitochondrial 2,4-dienoyl-CoA reductase

Chu, Xiaoyan; Yu, Wenhua; Chen, Gong; Ding, Li

doi:10.1016/s1046-5928(03)00191-8

Cited by 5 publications

(5 citation statements)

References 25 publications

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“…There is no obvious side chain in the active site to donate H ϩ directly to C␣, and we note that human mDECR has optimum activity near pH 6 (15). In many cases, (e.g.…”

Section: Structure Of Human Mitochondrial 24-dienoyl-coa Reductasementioning

confidence: 88%

“…Sample Preparation and Crystallization-The plasmid carrying the gene encoding truncated mDECR with an N-terminal His 6 tag was heat-shock transformed into a bacterial expression system (E. coli BL21(DE3), Novagen) and used according to established methods (15). To prepare SeMet mDECR the plasmid was transformed into the methionine auxotrophic strain of E. coli, B834(DE3) (Novagen), and selected on Luria-Bertani agar plates containing 100 g ml Ϫ1 ampicillin.…”

Section: Methodsmentioning

confidence: 99%

“…Expression and Purification of Human mDECR Wild-Type and Mutant Proteins-Established methods were again used to prepare the samples (15). The proteins, all observed with Ͼ95% purity by SDS-PAGE, were stored at Ϫ80°C in 50 mM potassium phosphate buffer, pH 7.5, 0.1 mM EDTA, 5% glycerol, and 5 mM ␤-mercaptoethanol.…”

Section: Methodsmentioning

confidence: 99%

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Structure and Reactivity of Human Mitochondrial 2,4-Dienoyl-CoA Reductase

Alphey

Byres

et al. 2005

Journal of Biological Chemistry

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Fatty acid catabolism by ␤-oxidation mainly occurs in mitochondria and to a lesser degree in peroxisomes. Polyunsaturated fatty acids are problematic for ␤-oxidation, because the enzymes directly involved are unable to process all the different double bond conformations and combinations that occur naturally. In mammals, three accessory proteins circumvent this problem by catalyzing specific isomerization and reduction reactions. Central to this process is the NADPH-dependent 2,4-dienoyl-CoA reductase. We present high resolution crystal structures of human mitochondrial 2,4-dienoylCoA reductase in binary complex with cofactor, and the ternary complex with NADP ؉ and substrate trans-2,trans-4-dienoyl-CoA at 2.1 and 1.75 Å resolution, respectively. The enzyme, a homotetramer, is a shortchain dehydrogenase/reductase with a distinctive catalytic center. Close structural similarity between the binary and ternary complexes suggests an absence of large conformational changes during binding and processing of substrate. The site of catalysis is relatively open and placed beside a flexible loop thereby allowing the enzyme to accommodate and process a wide range of fatty acids. Seven single mutants were constructed, by site-directed mutagenesis, to investigate the function of selected residues in the active site thought likely to either contribute to the architecture of the active site or to catalysis. The mutant proteins were overexpressed, purified to homogeneity, and then characterized. The structural and kinetic data are consistent and support a mechanism that derives one reducing equivalent from the cofactor, and one from solvent. Key to the acquisition of a solvent-derived proton is the orientation of substrate and stabilization of a dienolate intermediate by Tyr-199, Asn-148, and the oxidized nicotinamide.Essential fatty acids and derivatives that mammals acquire from exogenous sources or by endogenous biosynthesis fulfill critical functions in numerous metabolic pathways, in endocrine, and signaling processes (1, 2). Fatty acids also provide much of the cells energy following degradation in a sequence of four enzyme-catalyzed reactions termed the ␤-oxidation cycle (1, 3, 4), a highly exergonic metabolic process so named because oxidation occurs at C␤ of a fatty acyl-coenzyme A (CoA) 1 derivative preceding cleavage of the C␣-C␤ bond. The initial step leading into ␤-oxidation is fatty acid activation by formation of a thiolester bond with CoA in a reaction catalyzed by acyl-CoA synthetase. The oxidation of the C␣-C␤ bond produces an olefin, then hydration and oxidation produce a carbonyl group at C␤. The fourth step is cleavage of the ␤-keto ester in a reverse Claisen condensation resulting in a new acyl-CoA derivative truncated by two C atoms, which are used to produce acetyl-CoA. These reactions proceed until eventually only acetyl-CoA is produced. Extensive studies have afforded a very clear picture of structure, mechanism, and specificity of the four enzymes (acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-L-hydrox...

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“…There is no obvious side chain in the active site to donate H ϩ directly to C␣, and we note that human mDECR has optimum activity near pH 6 (15). In many cases, (e.g.…”

Section: Structure Of Human Mitochondrial 24-dienoyl-coa Reductasementioning

confidence: 88%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Structure and Reactivity of Human Mitochondrial 2,4-Dienoyl-CoA Reductase

Alphey

Byres

et al. 2005

Journal of Biological Chemistry

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“…Mammalian mitochondrial isoforms of DECR have been characterized at the nucleotide [6]–[8] and protein level [9]–[13], and the structure of the human 120 kD isoform has been recently solved [14]. Another mitochondrial isoform with a molecular mass of 60 kD has been partially purified, but it has not been characterized at the molecular level.…”

Section: Introductionmentioning

confidence: 99%

Mitochondrial 2,4-dienoyl-CoA Reductase Deficiency in Mice Results in Severe Hypoglycemia with Stress Intolerance and Unimpaired Ketogenesis

et al. 2009

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The mitochondrial β-oxidation system is one of the central metabolic pathways of energy metabolism in mammals. Enzyme defects in this pathway cause fatty acid oxidation disorders. To elucidate the role of 2,4-dienoyl-CoA reductase (DECR) as an auxiliary enzyme in the mitochondrial β-oxidation of unsaturated fatty acids, we created a DECR–deficient mouse line. In Decr−/− mice, the mitochondrial β-oxidation of unsaturated fatty acids with double bonds is expected to halt at the level of trans-2, cis/trans-4-dienoyl-CoA intermediates. In line with this expectation, fasted Decr−/− mice displayed increased serum acylcarnitines, especially decadienoylcarnitine, a product of the incomplete oxidation of linoleic acid (C18:2), urinary excretion of unsaturated dicarboxylic acids, and hepatic steatosis, wherein unsaturated fatty acids accumulate in liver triacylglycerols. Metabolically challenged Decr−/− mice turned on ketogenesis, but unexpectedly developed hypoglycemia. Induced expression of peroxisomal β-oxidation and microsomal ω-oxidation enzymes reflect the increased lipid load, whereas reduced mRNA levels of PGC-1α and CREB, as well as enzymes in the gluconeogenetic pathway, can contribute to stress-induced hypoglycemia. Furthermore, the thermogenic response was perturbed, as demonstrated by intolerance to acute cold exposure. This study highlights the necessity of DECR and the breakdown of unsaturated fatty acids in the transition of intermediary metabolism from the fed to the fasted state.

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“…The metabolism of unsaturated fatty acids usually requires auxiliary enzymes including Δ 3 - Δ 2 -enoyl-CoA isomerase, Δ 3,5 - Δ 2,4 -dienoyl-CoA isomerase, and 2,4-dienoyl-CoA reductase. The rate-limiting step of the above three enzymatic reactions is catalyzed by 2,4-dienoyl-CoA reductase, which has been studied in sufficient detail recently ( − ). Our study raises the question as to whether the dehydrogenase might function as an isomerase in vivo in conditions in which the activity of the isomerase is decreased.…”

Section: Discussionmentioning

confidence: 99%

Intrinsic Isomerase Activity of Medium-Chain Acyl-CoA Dehydrogenase

Zeng

2005

Biochemistry

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Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We found that the enzyme has intrinsic isomerase activity, which was confirmed using incubation followed with HPLC analysis. The isomerase activity of the enzyme was thoroughly characterized through studies of kinetics, substrate specificity, pH dependence, and enzyme inhibition. E376 mutants were constructed, and mutant enzymes were purified and characterized. It was shown that E376 is the catalytic residue for both dehydrogenase and isomerase activities of the enzyme. The isomerase activity of medium-chain acyl-CoA dehydrogenase is probably a spontaneous process driven by thermodynamic equilibrium with the formation of a conjugated structure after deprotonation of substrate alpha proton. The energy level of the transition state may be lowered by a stable dienolate intermediate, which gains further stabilization via charge transfer with the electron-deficient FAD cofactor of the enzyme. This raises the question as to whether the dehydrogenase might function as an isomerase in vivo in conditions in which the activity of the isomerase is decreased.

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Expression, purification, and characterization of His-tagged human mitochondrial 2,4-dienoyl-CoA reductase

Cited by 5 publications

References 25 publications

Structure and Reactivity of Human Mitochondrial 2,4-Dienoyl-CoA Reductase

Structure and Reactivity of Human Mitochondrial 2,4-Dienoyl-CoA Reductase

Mitochondrial 2,4-dienoyl-CoA Reductase Deficiency in Mice Results in Severe Hypoglycemia with Stress Intolerance and Unimpaired Ketogenesis

Intrinsic Isomerase Activity of Medium-Chain Acyl-CoA Dehydrogenase

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