Studies of 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. Sequence alignment indicates that there are five highly conserved acidic residues, one of which might act as a proton donor. We constructed five mutant expression plasmids of human mitochondrial 2,4-dienoyl-CoA reductase using site-directed mutagenesis. Mutant proteins were overexpressed in Escherichia coli and purified with a nickel metal affinity column. Studies of these mutant proteins were carried out, and the proton donor is likely to be E276. Three substrate analogs were synthesized and characterized. Two analogs, 2-fluoro-2,4-octadienoyl-CoA and 5-methyl-2,4-hexadienoyl-CoA, were substrates of the enzyme. Another analog, 3-furan-2-yl-acrylyl-CoA, was not a substrate, but a competitive inhibitor of the enzyme. These studies increased our understanding of human mitochondrial 2,4-dienoyl-CoA reductase.     

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.     

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.     

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. XII. Reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli

Incorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli (E. coli) was investigated. When trans-2,cis-4-decadienoyl-CoA was incubated with 4R- or 4S-[4-2H1]NADPH in the presence of purified 2,4-dienoyl-CoA reductase, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to its pyrrolidine amide. On the other hand, when the dienoyl-CoA was incubated in the presence of NADPH and the reductase in 2H2O, two deuterium atoms were incorporated: One deuterium atom was located at the C-4 position of trans-2-decenoate, and the other at the C-5 position. The UV and shorter wavelengths of the visible spectrum of the reductase solution revealed that the reductase contained flavin as a prosthetic group. Therefore it is considered that a hydrogen atom of NADPH was first transferred to the flavin moiety of the reductase, and then the hydrogen atom was rapidly exchanged for one in the medium before its direct transfer to the substrate.     

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.     

Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646.

An aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646 was purified 196-fold by a combination of Mono-Q, Reactive Green 19 agarose affinity, and hydroxyapatite chromatographies. The purified enzyme runs as a single band of 140 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass was estimated to be 163 +/- 3.8 kDa by gel filtration, indicating that this enzyme is a monomeric protein. The binding of the enzyme to Reactive Green 19 agarose was Mg2+ dependent. The binding capacity was estimated to be about 0.2 mg of Reactive Green agarose per ml in the presence of 10 mM MgCl2. This enzyme can catalyze the reduction of a wide range of aryl carboxylic acids, including substituted benzoic acids, phenyl-substituted aliphatic acids, heterocyclic carboxylic acids, and polyaromatic ring carboxylic acids, to produce the corresponding aldehydes. The Km values for benzoate, ATP, and NADPH were determined to be 645 +/- 75, 29.3 +/- 3.1, and 57.3 +/- 12.5 microM, respectively. The Vmax was determined to be 0.902 +/- 0.04 micromol/min/mg of protein. Km values for (S)-(+)-alpha-methyl-4-(2-methylpropyl)-benzeneacetic acid (ibuprofen) and its (R)-(-) isomer were determined to be 155 +/- 18 and 34.5 +/- 2.5 microM, respectively. The Vmax for the (S)-(+) and (R)-(-) isomers were 1.33 and 0.15 micromol/min/mg of protein, respectively. Anthranilic acid is a competitive inhibitor with benzoic acid as a substrate, with a Ki of 261 +/- 30 microM. The N-terminal and internal amino acid sequences of a 76-kDa peptide from limited alpha-chymotrypsin digestion were determined.     

Characterization of recombinant Dictyostelium discoideum sepiapterin reductase expressed in E. coli

A cDNA clone (SSC801) putatively encoding sepiapterin reductase (SR) was obtained from the expressed sequence tag clones of Dictyostelium discoideum. The cDNA sequence of 878 nucleotides constituted an ORF of 265 amino acid residues but was missing a few N-terminal residues. The deduced amino acid sequence showed 29.8% identity with mouse SR sequence and a molecular mass of 29,969 Da. The coding sequence was cloned in E. coli expression vector and overexpressed. The purified His-tag recombinant enzyme was confirmed to have the genuine activity of SR to produce tetrahydrobiopterin from 6-pyruvoyltetrahydropterin in a coupled assay with 6-pyruvoyltetrahydropterin synthase as well as dihydrobiopterin from sepiapterin. However, dictyopterin was not observed in our assay condition. The enzyme was also inhibited by N-acetylserotonin and to a lesser extent by melatonin. Km values for NADPH and sepiapterin were 51.8+/-2.7 microM and 40+/-2 microM, respectively. Vmax was determined as 0.14 micromol/min/mg of protein.     

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.     

Purification and characterization of maleylacetate reductase from Nocardioides simplex 3E utilizing phenoxyalcanoic herbicides 2,4-D and 2,4,5-T.

Maleylacetate reductase was isolated and purified from the Gram-positive strain Nocardioides simplex 3E which is able to utilize the phenoxyalcanoic herbicides 2,4-D and 2,4,5-T. Cells were grown on 2,4-D as the sole carbon source. The enzyme was purified by 380-fold with 3.0% yield. The purified maleylacetate reductase is a homodimer with subunit molecular mass of 37 kD. The enzyme required NADH as a cofactor; the Km for maleylacetate is 25 microM; Vmax (with NADH as cofactor) and kcat are 185 U/mg and 6845 min-1, respectively. The enzyme is very unstable; its pH and temperature optima are at 7.0-7.1 and 50 degrees C, respectively.     

Expression, purification, characterization and crystallization of a recombinant human cytosolic beta-glucosidase produced in insect cells

Human cytosolic beta-glucosidase is a monomeric enzyme that hydrolyzes various beta-d-glycosides and its real physiological role remains unclear. Here, we describe the production of this enzyme in Sf9 cells with a N-terminal 6x His tag. The production yield of the recombinant protein was in the 10 to 30 mg/l range. The protein was purified to homogeneity using two chromatographic steps, taking advantage of the 6x His tag in the first step, then using the physical and chemical properties of the protein for ionic exchange. Gel filtration analysis revealed that the protein is monomeric as expected. The kinetic parameters for 4-methylumbelliferyl beta-L-glucopyranoside, VM and KM, were measured (KM=32 microM and VM=157 micromol/h/mg at pH 7.0) and found similar to those reported for either the natural isolated enzyme or the recombinant protein expressed in COS7 cells (KM of 60-70 microM and 40 microM, respectively). Protein crystals were obtained and are now under structural investigations. In summary, we set up a heterologous expression system in Sf9 insect cells allowing the expression and production of large amounts of a pure active human protein, suitable for crystallographic studies.     

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.     

Purification, reconstitution, and steady-state kinetics of the trans-membrane 17 beta-hydroxysteroid dehydrogenase 2

Human membrane 17 beta-hydroxysteroid dehydrogenase 2 is an enzyme essential in the conversion of the highly active 17beta-hydroxysteroids into their inactive keto forms in a variety of tissues. 17 beta-hydroxysteroid dehydrogenase 2 with 6 consecutive histidines at its N terminus was expressed in Sf9 insect cells. This recombinant protein retained its biological activity and facilitated the enzyme purification and provided the most suitable form in our studies. Dodecyl-beta-D-maltoside was found to be the best detergent for the solubilization, purification, and reconstitution of this enzyme. The overexpressed integral membrane protein was purified with a high catalytic activity and a purity of more than 90% by nickel-chelated chromatography. For reconstitution, the purified protein was incorporated into dodecyl-beta-D-maltoside-destabilized liposomes prepared from l-alpha-phosphatidylcholine. The detergent was removed by adsorption onto polystyrene beads. The reconstituted enzyme had much higher stability and catalytic activity (2.6 micromol/min/mg of enzyme protein with estradiol) than the detergent-solubilized and purified protein (0.9 micromol/min/mg of enzyme protein with estradiol). The purified and reconstituted protein (with a 2-kDa His tag) was proved to be a homodimer, and its functional molecular mass was calculated to be 90.4 +/- 1.2 kDa based on glycerol gradient analytical ultracentrifugation and chemical cross-linking study. The kinetic studies demonstrated that 17 beta-hydroxysteroid dehydrogenase 2 was an NAD-preferring dehydrogenase with the K(m) of NAD being 110 +/- 10 microM and that of NADP 9600 +/- 100 microM using estradiol as substrate. The kinetic constants using estradiol, testosterone, dihydrotestosterone, and 20 alpha-dihydroprogesterone as substrates were also determined.     

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.     

Hexavalent chromium reduction by an actinomycete, Arthrobacter crystallopoietes ES 32

Environmental contamination by hexavalent chromium, Cr(VI), presents a serious public health problem. This study assessed the reduction of Cr(VI) by intact cells and a cell-free extract (CFE) of an actinomycete, Arthrobacter crystallopoietes (strain ES 32), isolated from soil contaminated with dichromate. Both intact cells and CFE of A. crystallopoietes, displayed substantial reduction of Cr(VI). Intact cells reduced about 90% of the Cr(VI) added within 12 h and Cr(VI) was almost completely reduced after 24 h. The K _M and V _max of Cr(VI) bioreduction by intact cells were 2.61 μM and 0.0142 μmol/min/mg protein, respectively. Cell-free chromate reductase of the A. crystallopoietes (ES 32) reduced hexavalent chromium at a K _M of 1.78 μM and a V _max of 0.096 μmol/min/mg protein. The rate constant (k) of chromate reduction was inversely related to Cr(VI) concentration and the half-life (t _1/2) of Cr(VI) reduction increased with increasing concentration. A. crystallopoietes produced a periplasmic chromate reductase that was stimulated by NADH. Results indicate that A. crystallopoietes ES 32 can be used to detoxify Cr(VI) in polluted sites, particularly in stressed environments.     

Purification and characterization of heme oxygenase from chick liver. Comparison of the avian and mammalian enzymes

A major inducible form of heme oxygenase (EC 1.14.99.3) was purified from liver microsomes of chicks pretreated with cadmium chloride. The purification involved solubilization of microsomes with Emulgen 913 and sodium cholate, followed by DEAE-Sephacel, carboxymethyl-cellulose (CM-52) and hydroxyapatite chromatography, and FPLC through Superose 6 and 12 columns operating in series. The final product gave a single band on silver-stained SDS/polyacrylamide gels (Mr = 33,000). Optimal conditions for measurement of activity of solubilized heme oxygenase were studied. In a reconstituted system containing purified heme oxygenase, NADPH-cytochrome reductase, biliverdin reductase and NADPH, the Km for free heme was 3.8 +/- 0.5 microM; for heme in the presence of bovine serum albumin (5 mol heme/3 mol albumin) the Km was 5.0 +/- 0.8 microM; and the Km for NADPH was 6.1 +/- 0.4 microM (all values mean +/- SD, n = 3). Oxygen concentration as low as 15 microM, with saturating concentrations of heme and NADPH, did not affect the reaction rate, indicating that the supply of oxygen is not involved in the physiological regulation of activity of the enzyme. The pH optimum of the reaction was 7.4; at 37 degrees C, the apparent Vmax was 580 +/- 44 nmol biliverdin.(mg protein)-1.min-1 and the molecular activity was 19.2 min-1. Biliverdin IXa was the sole biliverdin isomer formed. In the presence of purified biliverdin reductase, biliverdin was converted quantitatively to bilirubin. Addition of catalase to the reconstituted system decreased the breakdown of heme to non-biliverdin products and led to nearly stoichiometric conversion of heme to biliverdin. Activity of the enzyme in the reconstituted system was inhibited by metalloporphyrins in the following order of decreasing potency: tin mesoporphyrin greater than tin protoporphyrin greater than zinc protoporphyrin greater than manganese protoporphyrin greater than cobalt protoporphyrin. Protoporphyrin (3.3 or 6.6 microM) (and several other porphyrins) and metallic ions (100 microM) alone had little if any inhibitory effect, except for Hg2+ which inhibited by 67% at 10 microM and totally at 15 microM. Following partial cleavage, fragments of the purified enzyme were sequenced. Comparison of sequences to those derived from cDNA sequences for the major inducible rat and human heme oxygenase showed 69% and 76% similarities, respectively. The histidine residue at position 132 of rat heme oxygenase-1 and the residues (Lys128-Arg136) flanking His132 were conserved in all three enzymes, as well as in the corresponding portion of a fourth less highly similar rat enzyme, heme oxygenase-2.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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 (C_18:2), 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.     

Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity

Glutathione reductase (E C: 1.8.1.7; GR) was purified from rainbow trout (Oncorhynchus mykiss) liver, and some characteristics of the enzyme were investigated. The purification procedure consisted of four steps: preparation of homogenate, ammonium sulfate fractionation, affinity chromatography on 2',5'-ADP Sepharose-4B and gel filtration chromatography on Sephadex G-200. The enzyme, with a specific activity of 27.45 U/mg protein, was purified 1,654-fold with a yield of 41%. Optimal pH, stable pH, optimal temperature, optimum ionic strength, molecular mass, KM and Vmax values for GSSG and NADPH were also determined for the enzyme. In addition, Ki values and inhibition types were determined for GSH and NADP+. Additionally, inhibitory effects of metal ions (Cd+2, Cu+2, Pb+2, Hg+2, Fe+3 and Al+3) on glutathione reductase were investigated. Ki constants and IC50 values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I], respectively. IC50 values of Cd+2,Cu+2, Pb+2, Hg+2, Fe+3 and Al+3 were 0.0655, 0.082, 0.122, 0.509, 0.797 and 0.804 mM, and the Ki constants for Cd+2 and Cu+2 were 0.104+/-0.001, 0.117+/-0.001, respectively.     

Interaction of 3,4-dienoyl-CoA thioesters with medium chain acyl-CoA dehydrogenase: stereochemistry of inactivation of a flavoenzyme

The medium chain acyl-CoA dehydrogenase is rapidly inhibited by racemic 3,4-dienoyl-CoA derivatives with a stoichiometry of two molecules of racemate per enzyme flavin. Synthesis of R- and S-3,4-decadienoyl-CoA shows that the R-enantiomer is a potent, stoichiometric, inhibitor of the enzyme. alpha-Proton abstraction yields an enolate to oxidized flavin charge-transfer intermediate prior to adduct formation. The crystal structure of the reduced, inactive enzyme shows a single covalent bond linking the C-4 carbon of the 2,4-dienoyl-CoA moiety and the N5 locus of reduced flavin. The kinetics of reversal of adduct formation by release of the conjugated 2,4-diene were evaluated as a function of both acyl chain length and truncation of the CoA moiety. The adduct is most stable with medium chain length allenic inhibitors. However, the adducts with R-3,4-decadienoyl-pantetheine and -N-acetylcysteamine are some 9- and >100-fold more kinetically stable than the full-length CoA thioester. Crystal structures of these reduced enzyme species, determined to 2.4 A, suggest that the placement of H-bonds to the inhibitor carbonyl oxygen and the positioning of the catalytic base are important determinants of adduct stability. The S-3,4-decadienoyl-CoA is not a significant inhibitor of the medium chain dehydrogenase and does not form a detectable flavin adduct. However, the S-isomer is rapidly isomerized to the trans-trans-2,4-conjugated diene. Protein modeling studies suggest that the S-enantiomer cannot approach close enough to the isoalloxazine ring to form a flavin adduct, but can be facilely reprotonated by the catalytic base. These studies show that truncation of CoA thioesters may allow the design of unexpectedly potent lipophilic inhibitors of fatty acid oxidation.     

Bacterial expression and purification of the Arabidopsis NADPH-cytochrome P450 reductase ATR2

An N-terminally modified form of the Arabidopsis NADPH-cytochrome P450 ATR2 (ATR2mod) was expressed from the tactac promoter in Escherichia coli to obtain high yields of the enzyme. The N-terminal modification eliminates the predicted chloroplast transit peptide of ATR2 allowing for more efficient expression. ATR2mod was purified from membrane extracts using a 2',5'-ADP-agarose affinity column. The specific activity of the purified ATR2mod for cytochrome c reduction was 9.4 micromol min(-1) mg(-1) and the K(m) for cytochrome c reduction was 15 +/- 2 microM. The purified NADPH-cytochrome P450 reductase was able to support function of CYP79B2.