酿酒酵母中NAD激酶同功酶对偶数位不饱和脂肪酸β-氧化的影响

ATP-NAD激酶利用ATP,催化NAD磷酸化,生成NADP,而ATP-NADH激酶则催化NAD和NADH发生磷酸化。酿酒酵母细胞内存在三种NAD激酶同功酶Utr1p、Pos5p和Yef1p,它们都是ATP-NADH激酶,对细胞内NADP(H)的供应起到重要作用。酵母偶数位双键不饱和脂肪酸的β-氧化依赖于过氧化物酶体基质内的NADPH。通过构建NAD激酶基因的单、双基因缺失株,并验证它们和对照菌株对不饱和脂肪酸的氧化、利用能力,证实NAD激酶同功酶,尤其是Pos5p,对过氧化物酶体基质内NADP(H)的供应起着重要作用,并推测NADP可以从过氧化物酶体膜外转运至过氧化物酶体基质内。     

beta-Oxidation of polyunsaturated fatty acids by rat liver peroxisomes. A role for 2,4-dienoyl-coenzyme A reductase in peroxisomal beta-oxidation

beta-Oxidation of unsaturated fatty acids was studied with isolated solubilized or nonsolubilized peroxisomes or with perfused liver isolated from rats treated with clofibrate. gamma-Linolenic acid gave the higher rate of beta-oxidation, while arachidonic acid gave the slower rate of beta-oxidation. Other polyunsaturated fatty acids (including docosahexaenoic acid) were oxidized at rates which were similar to, or higher than, that observed with oleic acid. Experiments with 1-14C-labeled polyunsaturated fatty acids demonstrated that these are chain-shortened when incubated with nonsolubilized peroxisomes. Spectrophotometric investigation of solubilized peroxisomal incubations showed that 2,4-dienoyl-CoA esters accumulated during peroxisomal beta-oxidation of fatty acids possessing double bond(s) at even-numbered carbon atoms. beta-Oxidation of [1-14C]docosahexaenoic acid by isolated peroxisomes was markedly stimulated by added NADPH or isocitrate. This fatty acid also failed to cause acyl-CoA-dependent NADH generation with conditions of assay which facilitate this using other acyl-CoA esters. These findings suggest that 2,4-dienoyl-CoA reductase participation is essential during peroxisomal beta-oxidation if chain shortening is to proceed beyond a delta 4 double bond. Evidence obtained using arachidionoyl-CoA, [1-14C]arachidonic acid, and [5,6,8,9,11,12,14,15-3H]arachidonic acid suggests that peroxisomal beta-oxidation also can proceed beyond a double bond positioned at an odd-numbered carbon atom. Experiments with isolated perfused livers showed that polyunsaturated fatty acids also in the intact liver are substrates for peroxisomal beta-oxidation, as judged by increased levels of the catalase-H2O2 complex on infusion of polyunsaturated fatty acids.     

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 human PICD gene encodes a cytoplasmic and peroxisomal NADP(+)-dependent isocitrate dehydrogenase.

Human PICD was identified by homology probing the data base of expressed sequence tags with the protein sequence of Saccharomyces cerevisiae Idp3p, a peroxisomal NADP(+)-dependent isocitrate dehydrogenase. The human PICD cDNA contains a 1242-base pair open reading frame, and its deduced protein sequence is 59% identical to yeast Idp3p. Expression of PICD partially rescued the fatty acid growth defect of the yeast idp3 deletion mutant suggesting that PICD is functionally homologous to Idp3p. Kinetic studies on bacterially expressed PICD demonstrated that this enzyme catalyzed the oxidative decarboxylation of isocitrate to 2-oxoglutarate with a specific activity of 22.5 units/mg and that PICD displayed K(M) values of 76 microM for isocitrate and 112 microM for NADP(+). In subcellular fractionation experiments, we found PICD in both peroxisomes and cytoplasm of human and rat liver cells, with approximately 27% of total PICD protein associated with peroxisomes. The presence of PICD in mammalian peroxisomes suggests roles in the regeneration of NADPH for intraperoxisomal reductions, such as the conversion of 2, 4-dienoyl-CoAs to 3-enoyl-CoAs, as well as in peroxisomal reactions that consume 2-oxoglutarate, namely the alpha-hydroxylation of phytanic acid. As for cytoplasmic PICD, the phenotypes of patients with glucose-6-phosphate dehydrogenase deficiency (Luzzatto, L., and Mehta, A. (1995) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) Vol. 3, 7th Ed., pp. 3367-3398, McGraw-Hill Inc., New York) suggest that PICD serves a significant role in cytoplasmic NADPH production, particularly under conditions that do not favor the use of the hexose monophosphate shunt (Luzzatto et al.).     

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.     

Effect of growth hormone on fatty acid oxidation: growth hormone increases the activity of 2,4-dienoyl-CoA reductase in mitochondria

The effect of growth hormone on the beta-oxidation of saturated and unsaturated fatty acids was studied with mitochondria isolated from control rats, hypophysectomized rats, and hypophysectomized rats treated with growth hormone. Rates of respiration supported by polyunsaturated fatty acylcarnitines, in contrast to rates observed with palmitoylcarnitine or oleoylcarnitine, were slightly lower in hypophysectomized rats than in normal rats, but were higher in hypophysectomized rats treated with growth hormone. The effects were most pronounced with docosahexaenoylcarnitine, the substrate with the highest degree of unsaturation. Since uncoupling of mitochondria with 2,4-dinitrophenol resulted in lower rates of docosahexaenoylcarnitine-supported respiration, while substitution of ATP for ADP yielded higher rates, it appears that energy is required for the effective oxidation of polyunsaturated fatty acids. Growth hormone treatment of hypophysectomized rats caused a threefold increase in the activity of 2,4-dienoyl-CoA reductase or 4-enoyl-CoA reductase (EC 1.3.1.34) in mitochondria, but not in peroxisomes. The activities of other beta-oxidation enzymes remained virtually unchanged. Rates of acetoacetate formation from linolenoylcarnitine, but not from palmitoylcarnitine, were stimulated by glutamate in mitochondria from hypophysectomized rats and hypophysectomized rats treated with growth hormone. All data together lead to the conclusion that the mitochondrial oxidation of highly polyunsaturated fatty acids is limited by the availability of NADPH and the activity of 2,4-dienoyl-CoA reductase which is induced by growth hormone treatment.     

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.     

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.     

Beta-oxidation of polyunsaturated fatty acids in peroxisomes. Subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase activity in rat liver

The metabolism of the double bonds at the delta 3 position in fatty acids was studied in rat liver. Infusion of delta 3-trans-dodecenoic acid into isolated perfused liver and subcellular fractionation studies showed the presence of both peroxisomal and mitochondrial delta 3,delta 2-enoyl-CoA isomerase activity (EC 5.3.3.8). These findings together with the previous demonstration of peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34) [(1981) J. Biol. Chem. 256, 8259-8262] and D-3-OH-acyl-CoA epimerase (EC 5.1.2.3) [(1985) FEBS Lett. 185, 129-134] activities show that peroxisomes possess all the auxiliary enzymes required for the beta-oxidation of unsaturated fatty acids.     

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.     

The glyoxysomal beta-oxidation system in cucumber seedlings: identification of enzymes required for the degradation of unsaturated fatty acids

Fat-degrading cotyledons from cucumber seedlings were investigated with respect to the enzymes metabolizing cis-unsaturated fatty acids. Isolated glyoxysomes degrade linoleic acid, the dominating fatty acid in the storage tissue of the seed. Glyoxysomes were shown to be the sole intracellular site of enzymes responsible for the degradation of unsaturated fatty acids. All three auxiliary enzyme activities discussed for the degradation of polyunsaturated fatty acids, 2,4-dienoyl-CoA reductase, enoyl-CoA isomerase, and 3-hydroxyacyl-CoA epimerase were localized within the matrix of glyoxysomes. They were not found in mitochondria. Separation of glyoxysomal matrix proteins on CM-cellulose revealed that epimerase activity was attributable to the multifunctional protein and also to another protein which apparently exhibited no other beta-oxidation activity. Furthermore, on the basis of the high epimerase activity present in glyoxysomes compared to a much lower 2,4-dienoyl-CoA reductase activity, the metabolism of unsaturated fatty acids via delta 2-cis-enoyl-CoA is considered as alternative to the reductase-dependent pathway.     

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.     

Inhibitory effects of some long-chain unsaturated fatty acids on mitochondrial beta-oxidation. Effects of streptozotocin-induced diabetes on mitochondrial beta-oxidation of polyunsaturated fatty acids.

Evidence showing that some unsaturated fatty acids, and in particular docosahexaenoic acid, can be powerful inhibitors of mitochondrial beta-oxidation is presented. This inhibitory property is, however, also observed with the cis- and trans-isomers of the C18:1(16) acid. Hence it is probably the position of the double bond(s), and not the degree of unsaturation, which confers the inhibitory property. It is suggested that the inhibitory effect is caused by accumulation of 2,4-di- or 2,4,7-tri-enoyl-CoA esters in the mitochondrial matrix. This has previously been shown to occur with these fatty acids, in particular when the supply of NADPH was limiting 2,4-dienoyl-CoA reductase (EC 1.3.1.-) activity [Hiltunen, Osmundsen & Bremer (1983) Biochim. Biophys. Acta 752, 223-232]. Liver mitochondria from streptozotocin-diabetic rats showed an increased ability to beta-oxidize 2,4-dienoyl-CoA-requiring acylcarnitines. Docosahexaenoylcarnitine was also found to be less inhibitory at lower concentrations with incubation under coupled conditions. With uncoupling conditions there was little difference between mitochondria from normal and diabetic rats in these respects. This correlates with a 5-fold stimulation of 2,4-dienoyl-CoA reductase activity found in mitochondria from streptozotocin-diabetic rats.     

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.     

The known purified mammalian 2,4-dienoyl-CoA reductases are mitochondrial isoenzymes

The aim of this work was to determine the subcellular location of mammalian 2,4-dienoyl-CoA reductase, a key enzyme for degradation of polyunsaturated fatty acids by beta-oxidation. The enzyme was purified according to Kimura et al. (J Biochem 96:1463, 1984), and antibodies were raised in rabbits. Monospecific antibodies were obtained via purification on an affinity column. Immunoblotting of isolated rat liver mitochondria and peroxisomes with the monospecific reductase antibody showed that the antigen was located only in mitochondria. Immunocytochemical experiments with liver tissue, using the protein A-gold labeling technique, confirmed this result. The similarity of their characteristics suggests that the purified reductases described in the literature are the same isoenzyme. Consequently, since the rat enzyme was localized here to the mitochondria, purification and characterization of peroxisomal mammalian reductases remain to be achieved in the future. In addition, a significant induction also of mitochondrial reductase by clofibrate was observed in the immunoblotting experiments.     

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.     

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.     

Analysis of the beta-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Delta3,Delta2-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10-25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through beta-oxidation was more severely reduced in mutants devoid of Delta3,Delta2-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Delta3,Delta2-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.