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

Mitochondrial 2,4-dienoyl-CoA reductase is a key enzyme for the beta-oxidation of unsaturated fatty acids. The cDNA of the full-length human mitochondrial 2,4-dienoyl-CoA reductase was previously cloned as pUC18::DECR. PCR methodologies were used to subclone the genes encoding various truncated human mitochondrial 2,4-dienoyl-CoA reductases from pUC18::DECR with primers that were designed to add six continuous histidine codons to the 3' or 5' primer. The PCR products were inserted into pLM1 expression vectors and overexpressed in Escherichia coli. A highly active truncated soluble protein was expressed and purified with a nickel HiTrap chelating metal affinity column to apparent homogeneity based on Coomassie blue-stained SDS-PAGE. The molecular weight of the protein subunit was 34 kDa. The purified protein is highly stable at room temperature, which makes it potentially valuable for protein crystallization. KM of 26.5 +/- 3.8 microM for 2,4-hexadienoyl-CoA, KM of 6.22 +/- 2.0 microM for 2,4-decadienoyl-CoA, and KM of 60.5 +/- 19.7 microM for NADPH, as well as Vmax of 7.78 +/- 1.08 micromol/min/mg for 2,4-hexadienoyl-CoA and Vmax of 0.74 +/- 0.07 micromol/min/mg for 2,4-decadienoyl-CoA were determined on kinetic study of the purified protein. The one-step purification of the highly active human mitochondrial 2,4-dienoyl-CoA reductase will greatly facilitate further investigation of this enzyme through site-directed mutagenesis and enzyme catalyzed reactions with substrate analogs as well as protein crystallization for solving its three-dimensional structure.     

Structure and reactivity of human mitochondrial 2,4-dienoyl-CoA reductase: enzyme-ligand interactions in a distinctive short-chain reductase active site

Fatty acid catabolism by beta-oxidation mainly occurs in mitochondria and to a lesser degree in peroxisomes. Poly-unsaturated fatty acids are problematic for beta-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-dienoyl-CoA 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 A resolution, respectively. The enzyme, a homotetramer, is a short-chain 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.     

Purification by affinity chromatography of 2,4-dienoyl-CoA reductases from bovine liver and Escherichia coli

1. Dye-ligand chromatography using immobilized Cibacron blue F3GA (blue Sepharose CL-6B) and Procion red HE3B (Matrex gel red A) as matrices and general ligand chromatography employing immobilized 2',5'-ADP (2',5'-ADP-Sepharose 4B) and immobilized 3',5'-ADP (3',5'-ADP-Agarose) were employed for purification of NADPH-dependent 2-enoyl-CoA reductase and 2,4-dienoyl-CoA reductase from bovine liver (formerly called 4-enoyl-CoA reductase [Kunau, W. H. and Dommes, P. (1978) Eur. J. Biochem. 91, 533-544], as well as 2,4-dienoyl-CoA reductase from Escherichia coli. 2. The NADPH-dependent 2-enoyl-CoA reductase from bovine liver mitochondria was separated from 2,4-dienoyl-CoA reductase by dye-ligand chromatography (Matrex gel red A/KCl gradient) as well as by general ligand affinity chromatography (2',5'-ADP-Sepharose 4B/NADP gradient). The enzyme was obtained in a highly purified form. 3. The NADPH-dependent 2,4-dienoyl-CoA reductase from bovine liver mitochondria was purified to homogeneity using blue Sepharose CL-6B, Matrex gel red A, and 2',5'-ADP-Sepharose 4B chromatography. 4. The bacterial 2,4-dienoyl-CoA reductase was completely purified by ion-exchange chromatography on DEAE-cellulose followed by a single affinity chromatography step employing 2',5'-ADP-Sepharose 4B and biospecific elution from the column with a substrate, trans,trans-2,4-decadienoyl-CoA. 5. The application of dye-ligand and general ligand affinity chromatography for purification of NADPH-dependent 2,4-dienoyl-CoA reductases taking part in the beta-oxidation of unsaturated fatty acids is discussed. It is concluded that making use of coenzyme specificity for binding and substrate specificity for elution is essential for obtaining homogeneous enzyme preparations.     

Two distinct proton donors at the active site of Escherichia coli 2,4-dienoyl-CoA reductase are responsible for the formation of different products

NADPH-dependent 2,4-dienoyl-CoA reductase (DCR) is one of the auxiliary enzymes required for the beta-oxidation of unsaturated fatty acids. Mutants of Escherichia coli DCR were generated by site-directed mutagenesis to explore the molecular mechanism of this enzyme. The Tyr166Phe mutant, which was expected to be inactive due to the loss of its putative proton donor residue, exhibited 27% of the wild-type activity. However, the product of the reduction was 3-enoyl-CoA instead of 2-enoyl-CoA, the normal product. Glu164 seems to function as proton donor in the Tyr166Phe mutant, because the Tyr166Phe/ Glu164Gln double mutant was inactive whereas the Glu164Ala mutant exhibited low but significant activity. His252 is important for the efficient operation of Tyr166 because a His252Ala mutation by itself reduced the activity of DCR by 3 orders of magnitude, whereas the Tyr166Phe/His252Ala double mutation exhibited 4.4% of the wild-type activity. This data supports a mechanism that has Tyr166 with the assistance of His252 acting as proton donor in the wild-type enzyme to produce 2-enoyl-CoA, whereas Glu164 serves as the proton donor in the absence of Tyr166 to yield 3-enoyl-CoA. A Cys337Ala mutation, which resulted in the loss of most of the iron and acid-labile sulfur, decreased the reductase activity more than 1000-fold. This observation agrees with the proposed operation of an intramolecular electron transport chain that is essential for the effective catalysis of E. coli DCR.     

2,4-Dienoyl coenzyme A reductases from bovine liver and Escherichia coli. Comparison of properties

2,4-Dienoyl-CoA reductases, enzymes of the beta-oxidation of unsaturated fatty acids which were purified from bovine liver and oleate-induced cells of Escherichia coli, revealed very similar substrate specificities but distinctly different molecular properties. The subunit molecular weights, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 32,000 and 73,000 for the mammalian and the bacterial enzyme, respectively. The native molecular weights, calculated from sedimentation coefficients and Stokes radii yielded 124,000 for the bovine liver and 70,000 for the bacterial enzyme. Thus, bovine liver 2,4-dienoyl-CoA reductase is a tetramer consisting of four identical subunits. The E. coli 2,4-dienoyl-CoA reductase, however, possesses a monomeric structure. The latter enzyme contains 1 mol of FAD/mol of enzyme, whereas the former reductase is not a flavoprotein. The bovine liver reductase reduced 2-trans, 4-cis- and 2-trans,4-trans-decadienoyl-CoA to 3-trans-decenoyl-CoA. The E. coli reductase catalyzed the reduction of the same two substrates but in contrast yielded 2-trans-decenoyl-CoA as reaction product. Certain other properties of the two 2,4-dienoyl-CoA reductases are also presented. The localization of the reductase step within the degradation pathway of 4-cis-decenoyl-CoA, a metabolite of linoleic acid, is discussed.     

Cloning, expression, and purification of the functional 2,4-dienoyl-CoA reductase from rat liver mitochondria.

The mitochondrial 2,4-dienoyl-CoA reductase (EC 1.3.1.34) is an auxiliary enzyme for the beta-oxidation of unsaturated fatty acids. Import of this enzyme into the mitochondria requires a mitochondrial signal sequence at the amino terminus of the polypeptide chain which is processed/removed once inside the mitochondria. The cDNA of the full-length 2,4-dienoyl-CoA reductase was previously cloned as pRDR181. PCR methodologies were used to subclone the gene encoding the functional 2,4-dienoyl-CoA reductase from pRDR181. The PCR product was inserted into a pET15b expression vector and overexpressed in Escherichia coli. The soluble expressed protein can be separated into high- and low-activity fractions. The low-activity fraction can be converted to the high specific activity form by thermal annealing, suggesting it is a metastable misfolded form of the enzyme. Using ion-exchange and affinity chromatography, the enzyme has been purified to homogeneity and exhibits a single band on Coomassie blue-stained SDS-PAGE. The molecular mass of 32,413 Da determined by electrospray ionization-mass spectrometry indicates that the amino-terminal methionine had been removed. The Michaelis constants for trans-2, trans-4-hexadienoyl-CoA and NADPH were determined to be 0.46 and 2.5 microM, respectively; a turnover number of 2.1 s(-1) was calculated.     

cDNA cloning of rat liver 2,4-dienoyl-CoA reductase

cDNA clones of 2,4-dienoyl-CoA reductase were isolated from rat liver cDNA libraries constructed in phages lambda gt11 and lambda gt10. Hybrid selected translation analysis revealed that 2,4-dienoyl-CoA reductase was translated as a polypeptide with a molecular weight of about 36,000, which was about 3,000 molecular weight units larger than mature reductase. Sequencing analysis revealed that the open reading frame encoded a polypeptide consisting of 335 amino acid residues (predicted molecular weight = 36,132), which contained an N-terminal extension peptide of 34 amino acid residues (presequence) in addition to the mature enzyme. Thus, 2,4-dienoyl-CoA reductase is synthesized as a larger precursor polypeptide, and the N-terminal extension peptide may be acting as the mitochondrial import signal.     

The mechanism of dienoyl-CoA reduction by 2,4-dienoyl-CoA reductase is stepwise: observation of a dienolate intermediate

The chemical mechanism of the 2,4-dienoyl-CoA reductase (EC 1.3.1.34) from rat liver mitochondria has been investigated. This enzyme catalyzes the NADPH-dependent reduction of 2,4-dienoyl-coenzyme A (CoA) thiolesters to the resulting trans-3-enoyl-CoA. Steady-state kinetic parameters for trans-2,trans-4-hexadienoyl-CoA and 5-phenyl-trans-2,trans-4-pentadienoyl-CoA were determined and demonstrated that the dienoyl-CoA and NADPH bind to the 2,4-dienoyl-CoA reductase via a sequential kinetic mechanism. Kinetic isotope effect studies and the transient kinetics of substrate binding support a random order of nucleotide and dienoyl-CoA addition. The large normal solvent isotope effects on V/K ((D)(2)(O)V/K) and V ((D)(2)(O)V) for trans-2,trans-4-hexadienoyl-CoA reduction indicate that a proton transfer step is rate limiting for this substrate. The stability gained by conjugating the phenyl ring to the diene in PPD-CoA results in the reversal of the rate-determining step, as evidenced by the normal isotope effects on V/K(CoA) ((D)V/K(CoA)) and V/K(NADPH) ((D)V/K(NADPH)). The reversal of the rate-determining step was supported by transient kinetics where a burst was observed for the reduction of trans-2,trans-4-hexadienoyl-CoA but not for 5-phenyl-trans-2,trans-4-pentadienoyl-CoA reduction. The chemical mechanism is stepwise where hydride transfer from NADPH occurs followed by protonation of the observable dienolate intermediate, which has an absorbance maximum at 286 nm. The exchange of the C alpha protons of trans-3-decenoyl-CoA, catalyzed by the 2,4-dienoyl-CoA reductase, in the presence of NADP(+) suggests that formation of the dienolate is catalyzed by the enzyme active site.     

Studies on the metabolism of unsaturated fatty acids. XV. Purification and properties of 2,4-dienoyl-CoA reductase from rat liver peroxisomes

Peroxisomal 2,4-dienoyl-CoA reductase was purified from rat liver to homogeneity. The subunit molecular weight of 33,000 was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The native molecular weight close to 120,000 was estimated by gel filtration on Sephacryl S-300 Superfine. trans-2, trans-4-Decadienoyl-CoA was the most active substrate among the dienoyl-CoA's of various chain lengths. The total activity of peroxisomal 2,4-dienoyl-CoA reductase exceeded that of the mitochondrial one even in the livers of rats fed with a standard diet. Furthermore both reductases were remarkably and coordinately induced in the livers of clofibrate-treated rats.     

Evidence for the essential function of 2,4-dienoyl-coenzyme A reductase in the beta-oxidation of unsaturated fatty acids in vivo. Isolation and characterization of an Escherichia coli mutant with a defective 2,4-dienoyl-coenzyme A reductase

For the purpose of assessing in vivo the importance of 2,4-dienoyl-CoA reductase (EC 1.3.1.34) in the beta-oxidation of unsaturated fatty acids, reductase mutants of Escherichia coli were isolated by selecting cells that were able to grow on oleate but not on petroselinic acid (6-cis-octadecenoic acid). One mutant (fadH) exhibited 12% of the 2,4-dienoyl-CoA reductase activity present in the parental strain with other beta-oxidation enzymes being essentially unaffected. Antireductase antibodies were used to show that the mutant contains a fadH gene product at a level similar to that observed in the parental strain. Thus, the mutation seems to have resulted in the synthesis of a fadH gene product with lower specific activity. The mutation was mapped in the 71-75-min region of the E. coli chromosome where no other gene for beta-oxidation enzymes has so far been located. Complementation of the mutation by F'141, which carries the 67-75.5-min region of the E. coli genome, resulted in an increase in the 2,4-dienoyl-CoA reductase activity to 80% of the level found in the parental strain. Measurements of respiration with petroselinic acid as the substrate showed rates to be linearly dependent on the 2,4-dienoyl-CoA reductase activity up to levels found in wild-type E. coli. 2,4-Dienoyl-CoA reductase, like other enzymes of beta-oxidation, was induced when E. coli was grown on a long chain fatty acid as the sole carbon source. It is concluded that 2,4-dienoyl-CoA reductase is required in vivo for the beta-oxidation of unsaturated fatty acids with double bonds extending from even-numbered carbon atoms.     

2,4-Dienoyl-CoA reductase from Escherichia coli is a novel iron-sulfur flavoprotein that functions in fatty acid beta-oxidation

2,4-Dienoyl-CoA reductase is an enzyme that is required for the beta-oxidation of unsaturated fatty acids with even-numbered double bonds. The 2,4-dienoyl-CoA reductase from Escherichia coli was studied to explore the catalytic and structural properties that distinguish this enzyme from the corresponding eukaryotic reductases. The E. coli reductase was found to contain 1 mol of flavin mononucleotide and 4 mol each of acid-labile iron and sulfur in addition to 1 mol of flavin adenine dinucleotide per mole of protein. Redox titrations revealed a requirement for 5 mol of electrons to completely reduce 1 mol of enzyme and provided evidence for the formation of a red semiquinone intermediate. The reductase caused a significant polarization of the substrate carbonyl group as indicated by an enzyme-induced red shift of 38 nm in the spectrum of 5-phenyl-2,4-pentadienoyl-CoA. However, suspected cis --> trans isomerase and Delta(3),Delta(2)-enoyl-CoA isomerase activities were not detected in this enzyme. It is concluded that the 2, 4-dienoyl-CoA reductases from E. coli and eukaryotic organisms are structurally and mechanistically unrelated enzymes that catalyze the same type of reaction with similar efficiencies.     

3-Hydroxyacyl-CoA epimerases of rat liver peroxisomes and Escherichia coli function as auxiliary enzymes in the beta-oxidation of polyunsaturated fatty acids

The beta-oxidation of 2-trans,4-cis-decadienoyl-CoA, an assumed metabolite of linoleic acid, by purified enzymes from mitochondria, peroxisomes, and Escherichia coli was studied. 2-trans,4-cis-Decadienoyl-CoA is an extremely poor substrate of the beta-oxidation system reconstituted from mitochondrial enzymes. The results of a kinetic evaluation lead to the conclusion that in mitochondria 2-trans,4-cis-decadienoyl-CoA is not directly beta-oxidized, but instead is reduced by NADPH-dependent 2,4-dienoyl-CoA reductase prior to its beta-oxidation. Hence, the mitochondrial beta-oxidation of 2-trans,4-cis-decadienoyl-CoA does not require 3-hydroxyacyl-CoA epimerase, a conclusion which agrees with the finding that 3-hydroxyacyl-CoA epimerase is absent from mitochondria (Chu, C.-H., and Schulz, H. (1985) FEBS Lett. 185, 129-134). However, 2-trans,4-cis-decadienoyl-CoA can be slowly oxidized by the bifunctional beta-oxidation enzyme from rat liver peroxisomes, as well as by the fatty acid oxidation complex from E. coli. The observed rates of 2-trans,4-cis-decadienoyl-CoA degradation by these two multi-functional proteins were significantly higher than the values calculated according to steady-state velocity equations derived for coupled enzyme reactions. This is attributed to the direct transfer of L-3-hydroxy-4-cis-decenoyl-CoA from the active site of enoyl-CoA hydratase to that of 3-hydroxyacyl-CoA dehydrogenase on the same protein molecule. All observations together lead to the suggestion that the chain shortening of 2-trans,4-cis-decadienoyl-CoA in peroxisomes and in E. coli occurs simultaneously by two different pathways. The major pathway involves the NADPH-dependent 2,4-dienoyl-CoA reductase, whereas 3-hydroxyacyl-CoA epimerase functions in the metabolism of D-3-hydroxyoctanoyl-CoA which is formed via the minor pathway.     

Delta3,5,7,Delta2,4,6-trienoyl-CoA isomerase, a novel enzyme that functions in the beta-oxidation of polyunsaturated fatty acids with conjugated double bonds.

The mitochondrial metabolism of unsaturated fatty acids with conjugated double bonds at odd-numbered positions, e.g. 9-cis, 11-trans-octadecadienoic acid, was investigated. These fatty acids are substrates of beta-oxidation in isolated rat liver mitochondria and hence are expected to yield 5,7-dienoyl-CoA intermediates. 5, 7-Decadienoyl-CoA was used to study the degradation of these intermediates. After introduction of a 2-trans-double bond by acyl-CoA dehydrogenase or acyl-CoA oxidase, the resultant 2,5, 7-decatrienoyl-CoA can either continue its pass through the beta-oxidation cycle or be converted by Delta3,Delta2-enoyl-CoA isomerase to 3,5,7-decatrienoyl-CoA. The latter compound was isomerized by a novel enzyme, named Delta3,5,7,Delta2,4, 6-trienoyl-CoA isomerase, to 2,4,6-decatrienoyl-CoA, which is a substrate of 2,4-dienoyl-CoA reductase (Wang, H.-Y. and Schulz, H. (1989) Biochem. J. 264, 47-52) and hence can be completely degraded via beta-oxidation. Delta3,5,7,Delta2,4,6-Trienoyl-CoA isomerase was purified from pig heart to apparent homogeneity and found to be a component enzyme of Delta3,5,Delta2,4-dienoyl-CoA isomerase. Although the direct beta-oxidation of 2,5,7-decatrienoyl-CoA seems to be the major pathway, the degradation via 2,4,6-trienoyl-CoA makes a significant contribution to the total beta-oxidation of this intermediate.     

Studies on the metabolism of unsaturated fatty acids. XVI. Purification and general properties of 2,4-dienoyl-CoA reductase from Candida lipolytica

2,4-Dienoyl-CoA reductase has been purified to homogeneity from Candida lipolytica cultivated in the presence of linoleic acid. The native enzyme had a molecular weight close to 360,000 as estimated by gel filtration on Sepharose CL-4B, whereas the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 33,000. The purified 2,4-dienoyl-CoA reductase from C. lipolytica gave a single precipitin line with antibodies raised against the purified enzyme from C. lipolytica. The general properties of the 2,4-dienyl-CoA reductase from C. lipolytica were examined. The enzyme had optimal pH at 6.5 and was inactivated by heat treatment at 50 degrees C for 10 min. trans-2,trans-4-Octadienoyl-CoA was the most active substrate of the dienoyl-CoA esters examined.     

Peroxisomal beta-oxidation of polyunsaturated fatty acids in Saccharomyces cerevisiae: isocitrate dehydrogenase provides NADPH for reduction of double bonds at even positions.

The beta-oxidation of saturated fatty acids in Saccharomyces cerevisiae is confined exclusively to the peroxisomal compartment of the cell. Processing of mono- and polyunsaturated fatty acids with the double bond at an even position requires, in addition to the basic beta-oxidation machinery, the contribution of the NADPH-dependent enzyme 2,4-dienoyl-CoA reductase. Here we show by biochemical cell fractionation studies that this enzyme is a typical constituent of peroxisomes. As a consequence, the beta-oxidation of mono- and polyunsaturated fatty acids with double bonds at even positions requires stoichiometric amounts of intraperoxisomal NADPH. We suggest that NADP-dependent isocitrate dehydrogenase isoenzymes function in an NADP redox shuttle across the peroxisomal membrane to keep intraperoxisomal NADP reduced. This is based on the finding of a third NADP-dependent isocitrate dehydrogenase isoenzyme, Idp3p, next to the already known mitochondrial and cytosolic isoenzymes, which turned out to be present in the peroxisomal matrix. Our proposal is strongly supported by the observation that peroxisomal Idp3p is essential for growth on the unsaturated fatty acids arachidonic, linoleic and petroselinic acid, which require 2, 4-dienoyl-CoA reductase activity. On the other hand, growth on oleate which does not require 2,4-dienoyl-CoA reductase, and NADPH is completely normal in Deltaidp3 cells.     

Beta-oxidation of polyunsaturated fatty acids with conjugated double bonds. Mitochondrial metabolism of octa-2,4,6-trienoic acid.

The mitochondrial beta-oxidation of octa-2,4,6-trienoic acid was studied with the aim of elucidating the degradation of unsaturated fatty acids with conjugated double bonds. Octa-2,4,6-trienoic acid was found to be a respiratory substrate of coupled rat liver mitochondria, but not of rat heart mitochondria. Octa-2,4,6-trienoyl-CoA, the product of the inner-mitochondrial activation of the acid, was chemically synthesized and its degradation by purified enzymes of beta-oxidation was studied spectrophotometrically and by use of h.p.l.c. This compound is a substrate of NADPH-dependent 2,4-dienoyl-CoA reductase or 4-enoyl-CoA reductase (EC 1.3.1.34), which facilitates its further beta-oxidation. The product obtained after the NADPH-dependent reduction of octa-2,4,6-trienoyl-CoA and one round of beta-oxidation was hex-4-enoyl-CoA, which can be completely degraded via beta-oxidation. It is concluded that polyunsaturated fatty acids with two conjugated double bonds extending from even-numbered carbon atoms can be completely degraded via beta-oxidation because their presumed 2,4,6-trienoyl-CoA intermediates are substrates of 2,4-dienoyl-CoA reductase.     

The crystal structure and reaction mechanism of Escherichia coli 2,4-dienoyl-CoA reductase

Escherichia coli 2,4-dienoyl-CoA reductase is an iron-sulfur flavoenzyme required for the metabolism of unsaturated fatty acids with double bonds at even carbon positions. The enzyme contains FMN, FAD, and a 4Fe-4S cluster and exhibits sequence homology to another iron-sulfur flavoprotein, trimethylamine dehydrogenase. It also requires NADPH as an electron source, resulting in reduction of the C4-C5 double bond of the acyl chain of the CoA thioester substrate. The structure presented here of a ternary complex of E. coli 2,4-dienoyl-CoA reductase with NADP+ and a fatty acyl-CoA substrate reveals a possible mechanism for substrate reduction and provides details of a plausible electron transfer mechanism involving both flavins and the iron-sulfur cluster. The reaction is initiated by hydride transfer from NADPH to FAD, which in turn transfers electrons, one at a time, to FMN via the 4Fe-4S cluster. In the final stages of the reaction, the fully reduced FMN provides a hydride ion to the C5 atom of substrate, and Tyr-166 and His-252 are proposed to form a catalytic dyad that protonates the C4 atom of the substrate and complete the reaction. Inspection of the substrate binding pocket explains the relative promiscuity of the enzyme, catalyzing reduction of both 2-trans,4-cis- and 2-trans,4-trans-dienoyl-CoA thioesters.     

Purification and characterization of 2-enoyl-CoA reductase from bovine liver.

Mitochondrial 2-enoyl-CoA reductase from bovine liver was purified and characterized. A simple three-step purification was developed, involving ion-exchange chromatography to separate the bulk of the NADPH-dependent 2,4-dienoyl-CoA reductase, followed by chromatography on Blue Sepharose and adenosine 2',5'-bisphosphate-Sepharose. Homogeneous enzyme with a subunit Mr of 35 500 is obtained in 35% yield. The Mr of the native enzyme, determined by three different methods, yielded values that suggest that the enzyme is dimeric. NADPH is required as cofactor, and cannot be replaced by NADH. The activity of the purified enzyme towards 2-trans-double bonds in 2-monoene and 2,4-diene structures was investigated. 2-Enoyl-CoA reductase reduced the double bonds in a series of 2-trans-monoenoyl-CoA esters with different chain lengths, but did not exhibit significant activity towards 2-trans-double bonds of 2,4-dienoyl-CoA esters. This result is discussed in the light of analogous observations with enoyl-CoA hydratase.     

利用mRNA差异显示技术研究香菇发育相关基因

以香菇子实体不分化突变株和对照菌株L98411为材料,利用mRNA差异显示技术研究二者的基因表达差异,共分离到21条差异片段,其中12个分别与泛素、ATP合成酶、锌指蛋白、GTP结合蛋白、延伸因子g1线粒体前体、过氧化物酶、硫酯酶、类TrpB酶、2,4-二烯酰辅酶A还原酶、糖苷水解酶、热激蛋白、疏水蛋白等已知基因有较高的同源性,这表明香菇子实体分化发育是一个复杂的过程,涉及到胞内转运、转录调控、细胞分化、蛋白合成、各种代谢途径中的酶、抗逆反应等诸多途径的协同调控。