Atmospheric depositions affect the growth patterns of Scots pines (Pinus sylvestris L.)-a long-term cause-effect monitoring study using biomarkers.

Recording the causes, effects, and effect mechanisms of vegetation health is crucial to understand process-pattern interactions in ecosystem processes. NOX and SOX in the form of air pollution are both triggers and sources of vegetation health that can have an effect on the local or the global level and whose impacts need to be monitored. In this study, the growth patterns in Scots pines (Pinus sylvestris L.) were studied in the context of changing atmospheric depositions in the lowlands of north-eastern Germany. Under the influence of atmospheric sulfur (S) and nitrogen (N) depositions, pine stands showed temporal variations in their normal growth behavior. In such cases, the patterns of normal growth can be suppressed or accelerated. Pine stands which were influenced by high S deposition up until 1990 changed from suppressed growth to accelerated growth by decreasing S, but increasing N depositions between 1990 and 2003. The cause of these changes in pine growth patterns was imbalances in S and N nutrition, in particular, enrichments of sulfate, non-protein nitrogen or arginine, and finally, also imbalances and deficiencies in phosphorus, glucose, and adenosine triphosphate in the needles. Our long-term monitoring study shows that biochemical markers (traits) are crucial bioindicators for the qualitative and quantitative assessment of tree vitality and growth patterns in Scots pines. Furthermore, we were able to show that NOX and SOX depositions need to be monitored locally to be able to assess the local effects of biomolecular markers on the growth patterns in Scots pine stands.

Oleate beta-oxidation in yeast involves thioesterase but not Yor180c protein that is not a dienoyl-CoA isomerase.

The beta-oxidation of oleic acid in Saccharomyces cerevisiae (S. cerevisiae) was studied by comparing the growth of wild-type cells on oleic acid or palmitic acid with the growth of mutants that either had a deletion in the YOR180c (DCI1) gene reported to encode delta3,5,delta2,4-dienoyl-CoA isomerase (dienoyl-CoA isomerase) or in the PTE1 gene encoding peroxisomal thioesterase 1. Growth of wild-type cells was indistinguishable from that of YOR180c mutant cells on either palmitic acid or oleic acid, whereas the PTE1 mutant grew slower and to a lower density on oleic acid but not on palmitic acid. The identification of 3,5-tetradecadienoic acid in the medium of wild-type cells but not in the medium of the PTE1 mutant proves the operation of the thioesterase-dependent pathway of oleate beta-oxidation in S. cerevisiae. Dienoyl-CoA isomerase activity was very low in wild-type cells, fourfold higher in the YOR180c mutant, and not associated with purified Yor180c protein. These observations support the conclusion that the YOR180c gene does not encode dienoyl-CoA isomerase.

Mitochondrial long chain fatty acid beta-oxidation in man and mouse.

Several mouse models for mitochondrial fatty acid beta-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD(-/-)), very long chain acyl-CoA dehydrogenase knockout (VLCAD(-/-)) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.

Identification and characterization of Escherichia coli thioesterase III that functions in fatty acid beta-oxidation.

When Escherichia coli is grown on oleic acid as the sole carbon source, most of this fatty acid is completely degraded by beta-oxidation. However, approximately 10% of the oleic acid is only partially degraded to 3,5- cis-tetradecadienoyl-CoA, which is hydrolyzed to 3,5- cis-tetradecadienoic acid and released into the growth medium. An investigation of thioesterases involved in this novel pathway of beta-oxidation led to the identification of a new thioesterase (thioesterase III) that is induced by growth of E. coli on oleic acid. This enzyme was partially purified and identified as the ybaW gene product by mass spectrometric analysis of tryptic peptides. The ybaW gene, which has a putative consensus sequence for binding the fatty acid degradation repressor, was cloned and expressed in E. coli. Thioesterase III was shown to be a long-chain acyl-CoA thioesterase that is most active with 3,5-tetradecadienoyl-CoA, a minor metabolite of oleate beta-oxidation. Its substrate specificity and induction by fatty acids agree with its proposed function in the thioesterase-dependent pathway of beta-oxidation. Thioesterase III is proposed to hydrolyze metabolites of beta-oxidation that are resistant to further degradation and that would inhibit the flux through the pathway if they were allowed to accumulate.

A novel paradigm of fatty acid beta-oxidation exemplified by the thioesterase-dependent partial degradation of conjugated linoleic acid that fully supports growth of Escherichia coli.

An alternative pathway of beta-oxidation for unsaturated fatty acids was studied in Escherichia coli. 9- cis,11- trans-Octadecadienoic acid (conjugated linoleic acid), a potential substrate of this pathway, was shown to support growth of E. coli in the absence of any other carbon source. The identification of 3,5-dodecadienoic acid in the growth medium revealed the partial beta-oxidation of conjugated linoleic acid to 3,5-dodecadienoyl-CoA, which was hydrolyzed to 3,5-dodecadienoic acid and released from cells. The involvement of acyl-CoA thioesterases in this process was evaluated by determining the substrate specificity of thioesterase II and comparing it with that of a novel thioesterase (thioesterase III) and by assessing mutant strains devoid of one or both of these thioesterases for growth on conjugated linoleic acid. Both thioesterases were highly active with 3,5-dodecadienoyl-CoA as substrate. A deficiency of either thioesterase decreased the growth rate of cells on conjugated linoleic acid but not on palmitic acid. The absence of both thioesterases reduced the cellular growth in a cumulative manner but did not abolish it. It is concluded that thioesterases II and III and at least one other thioesterase function in the partial degradation of conjugated linoleic acid via the thioesterase-dependent pathway of beta-oxidation, which provides all energy and carbon precursors required for the growth of E. coli.

Comparison of leaching tests to determine and quantify the release of inorganic contaminants in demolition waste.

The changes in waste management policy caused by the massive generation of waste materials (e.g. construction and demolition waste material, municipal waste incineration products) has led to an increase in the reuse and recycling of waste materials. For environmental risk assessment, test procedures are necessary to examine waste materials before they can be reused. In this article, results of column and lysimeter leaching tests having been applied to inorganic compounds in a reference demolition waste material are presented. The results show a good agreement between the leaching behaviour determined with the lysimeter unit and the column units used in the laboratory. In view of less time and system requirements compared to lysimeter systems, laboratory column units can be considered as a practicable instrument to assess the time-dependent release of inorganic compounds under conditions similar to those encountered in a natural environment. The high concentrations of elements in the seepage water at the initial stage of elution are reflected by the laboratory column leaching tests. In particular, authorities or laboratories might benefit and have an easy-to-use, but nevertheless reliable, method to serve as a basis for decision-making.

Multiple functions of type 10 17beta-hydroxysteroid dehydrogenase.

Human 17beta-hydroxysteroid dehydrogenase type 10 (17beta-HSD10) is a mitochondrial enzyme encoded by the SCHAD gene, which escapes chromosome X inactivation. 17Beta-HSD10/SCHAD mutations cause a spectrum of clinical conditions, from mild mental retardation to progressive infantile neurodegeneration. 17Beta-HSD10/SCHAD is essential for the metabolism of isoleucine and branched-chain fatty acids. It can inactivate 17beta-estradiol and steroid modulators of GABA(A) receptors, and convert 5alpha-androstanediol into 5alpha-dihydrotestosterone (DHT). Certain malignant prostatic epithelial cells contain high levels of 17beta-HSD10, generating 5alpha-DHT in the absence of testosterone. 17Beta-HSD10 has an affinity for amyloid-beta peptide, and might be linked to the mitochondrial dysfunction seen in Alzheimer's disease. This versatile enzyme might provide a new drug target for neuronal excitability control and for intervention in Alzheimer's disease and certain cancers.

Domain swapping in the low-similarity isomerase/hydratase superfamily: the crystal structure of rat mitochondrial Delta3, Delta2-enoyl-CoA isomerase.

Two monofunctional Delta(3), Delta(2)-enoyl-CoA isomerases, one in mitochondria (mECI) and the other in both mitochondria and peroxisomes (pECI), belong to the low-similarity isomerase/hydratase superfamily. Both enzymes catalyze the movement of a double bond from C3 to C2 of an unsaturated acyl-CoA substrate for re-entry into the beta-oxidation pathway. Mutagenesis has shown that Glu165 of rat mECI is involved in catalysis; however, the putative catalytic residue in yeast pECI, Glu158, is not conserved in mECI. To elucidate whether Glu165 of mECI is correctly positioned for catalysis, the crystal structure of rat mECI has been solved. Crystal packing suggests the enzyme is trimeric, in contrast to other members of the superfamily, which appear crystallographically to be dimers of trimers. The polypeptide fold of mECI, like pECI, belongs to a subset of this superfamily in which the C-terminal domain of a given monomer interacts with its own N-terminal domain. This differs from that of crotonase and 1,4-dihydroxy-2-naphtoyl-CoA synthase, whose C-terminal domains are involved in domain swapping with an adjacent monomer. The structure confirms Glu165 as the putative catalytic acid/base, positioned to abstract the pro-R proton from C2 and reprotonate at C4 of the acyl chain. The large tunnel-shaped active site cavity observed in the mECI structure explains the relative substrate promiscuity in acyl-chain length and stereochemistry. Comparison with the crystal structure of pECI suggests the catalytic residues from both enzymes are spatially conserved but not in their primary structures, providing a powerful reminder of how catalytic residues cannot be determined solely by sequence alignments.

3-Hydroxyacyl-CoA dehydrogenase and short chain 3-hydroxyacyl-CoA dehydrogenase in human health and disease.

3-Hydroxyacyl-CoA dehydrogenase (HAD) functions in mitochondrial fatty acid beta-oxidation by catalyzing the oxidation of straight chain 3-hydroxyacyl-CoAs. HAD has a preference for medium chain substrates, whereas short chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) acts on a wide spectrum of substrates, including steroids, cholic acids, and fatty acids, with a preference for short chain methyl-branched acyl-CoAs. Therefore, HAD should not be referred to as SCHAD. SCHAD is not a member of the HAD family, but instead, belongs to the short chain dehydrogenase/reductase superfamily. Previously reported cases of SCHAD deficiency are due to an inherited HAD deficiency. SCHAD, also known as 17beta-hydroxysteroid dehydrogenase type 10, is important in brain development and aging. Abnormal levels of SCHAD in certain brain regions may contribute to the pathogenesis of some neural disorders. The human SCHAD gene and its protein product, SCHAD, are potential targets for intervention in conditions, such as Alzheimer's disease, Parkinson's disease, and an X-linked mental retardation, that may arise from the impaired degradation of branched chain fatty acid and isoleucine.

Type 10 17beta-hydroxysteroid dehydrogenase catalyzing the oxidation of steroid modulators of gamma-aminobutyric acid type A receptors.

The steroids allopregnanolone and allotetrahydrodeoxycorticosterone (3alpha,5alpha-THDOC) are positive allosteric modulators of GABA(A) receptors, generated by the reduction of 5alpha-dihydroprogesterone (5alpha-DHP) and 5alpha-DHDOC, respectively, under the catalysis of human type 3 3alpha-hydroxysteroid dehydrogenase (HSD). However, brain enzymes catalyzing the conversion of such tetrahydrosteroids back to the corresponding 5alpha-dihydrosteroids remain to be identified. Characterization of human type 10 17beta-HSD provides a new insight into its importance for the oxidation of steroid modulators of GABA(A) receptors. The apparent catalytic efficiency (k(cat)/K(m)) of this enzyme for the oxidation of allopregnanolone and 3alpha,5alpha-THDOC are 432 and 1381 min(-1) mM(-1), respectively. This enzyme has negligible 3-ketosteroid reductase activity for 5alpha-DHP and 5alpha-DHDOC even in an acidic environment. Immunoreactivity against 17beta-HSD10 was found in a number of neuronal populations. Taken together, evidence suggests that 17beta-HSD10 is the brain enzyme capable of catalyzing the oxidation of steroid modulators of GABA(A) receptors.

Abundant type 10 17 beta-hydroxysteroid dehydrogenase in the hippocampus of mouse Alzheimer's disease model.

A full-length cDNA of mouse type 10 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD10) was cloned from brain, representing the accurate nucleotide sequence information that rendered possible an accurate deduction of the amino acid sequence of the wild-type enzyme. A comparison of sequences and three-dimensional models of this enzyme revealed that structures previously reported by other groups carry either a truncated or mutated amino-terminal sequence. Fusion of the first 11 residues of the wild-type enzyme to the green fluorescent protein directed the reporter protein into mitochondria. Thus, the N-terminus was identified as a mitochondrial targeting signal that accounts for the intracellular localization of the mouse enzyme. This enzyme is normally associated with mitochondria, not with the endoplasmic reticulum as suggested by its trivial name 'endoplasmic reticulum-associated amyloid-beta biding protein (ERAB)'. After its C-terminal region was used to raise rabbit anti-17 betaHSD10 antibodies, immunogold electron microscopy showed that an abundance of this enzyme could be found in hippocampal synaptic mitochondria of betaAPP transgenic mice, but not in normal controls. High levels of this enzyme may disrupt steroid hormone homeostasis in synapses and contribute to synapse loss in the hippocampus of the mouse Alzheimer's disease model.

Oxidative 3alpha-hydroxysteroid dehydrogenase activity of human type 10 17beta-hydroxysteroid dehydrogenase.

In vitro enzyme assays have demonstrated that human type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10) catalyzes the oxidation of 5alpha-androstane-3alpha,17beta-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17beta-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17beta-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17beta-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17beta-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3alpha-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17beta-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.

Accumulation of aluminum in rat brain: does it lead to behavioral and electrophysiological changes?

The present study was undertaken to examine possible aluminum (Al) accumulation in the brain of rats and to investigate whether subchronic exposure to the metal leads to behavioral and neurophysiological changes in both treated and control groups. Each of the groups consisted of 10 animals. Aluminum chloride (AlCl3) at a low (50 mg/kg/d) or high (200 mg/kg/d) dose was applied to male Wistar rats by gavage for 8 wk. Al-free water by gavage was given to the control group throughout the experiment. Behavioral effects were evaluated by open-field (OF) motor activity and by acoustic startle response (ASR). Electrophysiological examination was done by recording spontaneous activity and sensory-evoked potentials from the visual, somatosensory, as well as auditory cortex. The Al content of each whole brain was determined by electrothermal atomic absorption spectrophotometry. Subchronic Al exposure slightly caused some changes in the evoked potentials and electrocorticograms and in the OF and ASR performance, but these results were not statistically significant. The brain Al levels of the control and the low and high dose of Al-exposed groups were measured as 0.717+/-0.208 microg/g (wet weight), 0.963+/-0.491 microg/g (wet weight) and 1.816+/-1.157 microg/g (wet weight), respectively.

Effect of raw humus under two adult Scots pine stands on ectomycorrhization, nutritional status, nitrogen uptake, phosphorus uptake and growth of Pinus sylvestris seedlings.

Ectomycorrhiza (EM) formation improves tree growth and nutrient acquisition, particularly that of nitrogen (N). Few studies have coupled the effects of naturally occurring EM morphotypes to the nutrition of host trees. To investigate this, pine seedlings were grown on raw humus substrates collected at two forest sites, R2 and R3. Ectomycorrhiza morphotypes were identified, and their respective N uptake rates from organic (2-(13)C, (15)N-glycine) and inorganic ((15)NH(4)Cl, Na(15)NO(3), (15)NH(4)NO(3), NH(4)(15)NO(3)) sources as well as their phosphate uptake rates were determined. Subsequently, the growth and nutritional status of the seedlings were analyzed. Two dominant EM morphotypes displayed significantly different mycorrhization rates in the two substrates. Rhizopogon luteolus Fr. (RL) was dominant in R2 and Suillus bovinus (Pers.) Kuntze (SB) was dominant in R3. (15)N uptake of RL EM was at all times higher than that of SB EM. Phosphate uptake rates by the EM morphotypes did not differ significantly. The number of RL EM correlated negatively and the number of SB EM correlated positively with pine growth rate. Increased arginine concentrations and critical P/N ratios in needles indicated nutrient imbalances of pine seedlings from humus R2, predominantly mycorrhizal with RL. We conclude that different N supply in raw humus under Scots pine stands can induce shifts in the EM frequency of pine seedlings, and this may lead to EM formation by fungal strains with different ability to support tree growth.

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.

Large sulfur bacteria and the formation of phosphorite.

Phosphorite deposits in marine sediments are a long-term sink for an essential nutrient, phosphorus. Here we show that apatite abundance in sediments on the Namibian shelf correlates with the abundance and activity of the giant sulfur bacterium Thiomargarita namibiensis, which suggests that sulfur bacteria drive phosphogenesis. Sediments populated by Thiomargarita showed sharp peaks of pore water phosphate (/=50 grams of phosphorus per kilogram). Laboratory experiments revealed that under anoxic conditions, Thiomargarita released enough phosphate to account for the precipitation of hydroxyapatite observed in the environment.

Metabolic functions of the two pathways of oleate beta-oxidation double bond metabolism during the beta-oxidation of oleic acid in rat heart mitochondria.

Unsaturated fatty acids with odd-numbered double bonds, e.g. oleic acid, can be degraded by beta-oxidation via the isomerase-dependent pathway or the reductase-dependent pathway that differ with respect to the metabolism of the double bond. In an attempt to elucidate the metabolic functions of the two pathways and to determine their contributions to the beta-oxidation of unsaturated fatty acids, the degradation of 2-trans,5-cis-tetradecadienoyl-CoA, a metabolite of oleic acid, was studied with rat heart mitochondria. Kinetic measurements of metabolite and cofactor formation demonstrated that more than 80% of oleate beta-oxidation occurs via the classical isomerase-dependent pathway whereas the more recently discovered reductase-dependent pathway is the minor pathway. However, the reductase-dependent pathway is indispensable for the degradation of 3,5-cis-tetradecadienoyl-CoA, which is formed from 2-trans,5-cis-tetradecadienoyl-CoA by delta(3),delta(2)-enoyl-CoA isomerase, the auxiliary enzyme that is essential for the operation of the major pathway of oleate beta-oxidation. The degradation of 3,5-cis-tetradecadienoyl-CoA is limited by the capacity of 2,4-dienoyl-CoA reductase to reduce 2-trans,4-trans-tetradecadienoyl-CoA, which is rapidly formed from its 3,5 isomer by delta(3,5),delta(2,4)-dienoyl-CoA isomerase. It is concluded that both pathways are essential for the degradation of unsaturated fatty acids with odd-numbered double bonds inasmuch as the isomerase-dependent pathway facilitates the major flux through beta-oxidation and the reductase-dependent pathway prevents the accumulation of an otherwise undegradable metabolite.

Leaky beta-oxidation of a trans-fatty acid: incomplete beta-oxidation of elaidic acid is due to the accumulation of 5-trans-tetradecenoyl-CoA and its hydrolysis and conversion to 5-trans-tetradecenoylcarnitine in the matrix of rat mitochondria.

The degradation of elaidic acid (9-trans-octadecenoic acid), oleic acid, and stearic acid by rat mitochondria was studied to determine whether the presence of a trans double bond in place of a cis double bond or no double bond affects beta-oxidation. Rat mitochondria from liver or heart effectively degraded the coenzyme A derivatives of all three fatty acids. However, with elaidoyl-CoA as a substrate, a major metabolite accumulated in the mitochondrial matrix. This metabolite was isolated and identified as 5-trans-tetradecenoyl-CoA. In contrast, little or none of the corresponding metabolites were detected with oleoyl-CoA or stearoyl-CoA as substrates. A kinetic study of long-chain acyl-CoA dehydrogenase (LCAD) and very long-chain acyl-CoA dehydrogenase revealed that 5-trans-tetradecenoyl-CoA is a poorer substrate of LCAD than is 5-cis-tetradecenoyl-CoA, while both unsaturated acyl-CoAs are poor substrates of very long-chain acyl-CoA dehydrogenase when compared with myristoyl-CoA. Tetradecenoic acid and tetradecenoylcarnitine were detected by gas chromatography/mass spectrometry and tandem mass spectrometry, respectively, when rat liver mitochondria were incubated with elaidoyl-CoA but not when oleoyl-CoA was the substrate. These observations support the conclusion that 5-trans-tetradecenoyl-CoA accumulates in the mitochondrial matrix, because it is less efficiently dehydrogenated by LCAD than is its cis isomer and that the accumulation of this beta-oxidation intermediate facilitates its hydrolysis and conversion to 5-trans-tetradecenoylcarnitine thereby permitting a partially degraded fatty acid to escape from mitochondria. Analysis of this compromised but functional process provides insight into the operation of beta-oxidation in intact mitochondria.

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.

Functional characterization of Delta3,Delta2-enoyl-CoA isomerases from rat liver.

The degradation of unsaturated fatty acids by beta-oxidation involves Delta(3),Delta(2)-enoyl-CoA isomerases (enoyl-CoA isomerases) that catalyze 3-cis --> 2-trans and 3-trans --> 2-trans isomerizations of enoyl-CoAs and the 2,5 --> 3,5 isomerization of dienoyl-CoAs. An analysis of rat liver enoyl-CoA isomerases revealed the presence of a monofunctional enoyl-CoA isomerase (ECI) in addition to mitochondrial enoyl-CoA isomerase (MECI) in mitochondria, whereas peroxisomes contain ECI and multifunctional enzyme 1 (MFE1). Thus ECI, which previously had been described as peroxisomal enoyl-CoA isomerase, was found to be present in both peroxisomes and mitochondria. This enzyme seems to be identical with mitochondrial long-chain enoyl-CoA isomerase (Kilponen, J.M., Palosaari, P.M., and Hiltunen, J.K. 1990. Biochem. J. 269, 223-226). All three hepatic enoyl-CoA isomerases have broad chain length specificities but are distinguishable by their preferences for one of the three isomerization reactions. MECI is most active in catalyzing the 3-cis --> 2-trans isomerization; ECI has a preference for the 3-trans --> 2-trans isomerization, and MFE1 is the optimal isomerase for the 2,5 --> 3,5 isomerization. A functional characterization based on substrate specificities and total enoyl-CoA isomerase activities in rat liver leads to the conclusion that the 3-cis --> 2-trans and 2,5 --> 3,5 isomerizations in mitochondria are catalyzed overwhelmingly by MECI, whereas ECI contributes significantly to the 3-trans --> 2-trans isomerization. In peroxisomes, ECI is predicted to be the dominant enzyme for the 3-cis --> 2-trans and 3-trans --> 2-trans isomerizations of long-chain intermediates, whereas MFE1 is the key enzyme in the 2,5 --> 3,5 isomerization.