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The (3R)-hydroxyacyl-ACP dehydratase HadAB, involved in the biosynthetic pathway for mycolic acid (MA) of Mycobacterium tuberculosis, catalyzes the third step in the fatty acid (FA) elongation cycle, which is an ideal and actual target for anti-tubercular agent. Though HadAB is predicted to be a member of the hotdog superfamily, it shares no sequence identity with typical hotdog fold isoenzyme FabZ. To characterize the significance of HadAB from the perspective of structural biology, large amount of pure HadAB complex is required for biochemical characterization and crystallization. Here, we used a unique expression and purification method. HadA and HadB were cloned separately and co-expressed in Escherichia coli. After GST affinity chromatography, two steps of anion exchange chromatography and gel filtration, the purity of the protein as estimated by SDS-PAGE was >95%. Using hanging-drop vapor-diffusion method, crystals were obtained and diffracted X-rays to 1.75Å resolution. The crystal belongs to space group P41212, with unit-cell parameters a=b=82.0Å, c=139.8Å, α=β=γ=90.0°., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

A polyhydroxyalkanoate (PHA) was enzymatically synthesized in vitro, and the end structure of PHA associated with a chain transfer (CT) reaction was investigated. In the CT reaction, PHA chain transfers from PHA synthase (PhaC) to a CT agent, resulting in covalent bonding of CT agent to the PHA chain at its carboxyl end. In vitro CT reaction has never been demonstrated because of relatively low yields of in vitro synthesized poly[(R)-3-hydroxybutyrate)] (P(3HB)), which makes it difficult to characterize the end structures of the polymers by nuclear magnetic resonance (NMR). To overcome these difficulties, a novel in vitro synthesis method that produced relatively larger amounts of P(3HB) was developed by employing PhaCDa from Delftia acidovorans and two enantioselective enoyl-coenzyme A (CoA) hydratases which were R-hydratase (PhaJAc) from Aeromonas caviae and S-hydratase (FadB1x) from Pseudomonas putida KT2440 with β-butyrolactone and CoA as starting materials. Using this method, P(3HB) synthesis was performed with tetraethylene glycols (TEGs) as a discriminable CT agent, and the resultant P(3HB) was characterized by (1)H-NMR. NMR analysis revealed that the carboxylic end of P(3HB) was covalently linked to TEGs, providing the first direct evidence of in vitro CT reaction.

The (R)-specific enoyl-CoA hydratase gene (phaJ(HS21)) from Pseudomonas chlororaphis HS21 was overexpressed in various Pseudomonas strains, alone and in combination with the polyhydroxyalkanoate synthase gene (phaC(HS21)), for the biosynthesis of polyhydroxyalkanoates (PHAs) of altered monomer composition. Recombinant Pseudomonas strains harboring phaC(HS21) and phaJ(HS21) generated saturated and unsaturated monomers of C12-C14 in their PHAs. In particular, the level of the 3-hydroxytetradecenoate monomer in recombinant P. chlororaphis HS21 increased by approximately 260%. PhaJ(HS21) is expected to be useful in the biosynthesis of PHAs consisting of unusual monomer units.

We investigated the expression of (R)-specific enoyl coenzyme A hydratase (PhaJ) in Pseudomonas putida KT2440 accumulating polyhydroxyalkanoate (PHA) from sodium octanoate in order to identify biosynthesis pathways of PHAs from fatty acids in pseudomonads. From a database search through the P. putida KT2440 genome, an additional phaJ gene homologous to phaJ4(Pa) from Pseudomonas aeruginosa, termed phaJ4(Pp), was identified. The gene products of phaJ1(Pp), which was identified previously, and phaJ4(Pp) were confirmed to be functional in recombinant Escherichia coli on PHA synthesis from sodium dodecanoate. Cytosolic proteins from P. putida grown on sodium octanoate were subjected to anion exchange chromatography and one of the eluted fractions with hydratase activity included PhaJ4(Pp), as revealed by western blot analysis. These results strongly suggest that PhaJ4(Pp) forms a channeling route from β-oxidation to PHA biosynthesis in P. putida. Moreover, the substrate specificity of PhaJ1(Pp) was suggested to be different from that of PhaJ1(Pa) from P. aeruginosa although these two proteins share 67% amino acid sequence identity.

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO(2) fixation in various Crenarchaea, as studied in some detail in Metallosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.

The assimilation of aromatic compounds by microbial species requires specialized enzymes to cleave the thermodynamically stable ring. In the recently discovered benzoate-oxidation (box) pathway in Burkholderia xenovorans LB400, this is accomplished by a novel dihydrodiol lyase (BoxC(C)). Sequence analysis suggests that BoxC(C) is part of the crotonase superfamily but includes an additional uncharacterized region of approximately 115 residues that is predicted to mediate ring cleavage. Processing of X-ray diffraction data to 1.5 A resolution revealed that BoxC(C) crystallized with two molecules in the asymmetric unit of the P2(1)2(1)2(1) space group, with a solvent content of 47% and a Matthews coefficient of 2.32 A(3) Da(-1). Selenomethionine BoxC(C) has been purified and crystals are currently being refined for anomalous dispersion studies.

Enzymes involved in carnitine metabolism of Proteus sp. are encoded by the cai genes organised as the caiTABCDEF operon. The complete operon could be sequenced from the genomic DNA of Proteus sp. Amino acid sequence similarities and/or enzymatic analysis confirmed the function assigned to each protein involved in carnitine metabolism. CaiT was suggested to be an integral membrane protein responsible for the transport of betaines. The caiA gene product was shown to be a crotonobetainyl-CoA reductase catalysing the irreversible reduction of crotonobetainyl-CoA to gamma-butyrobetainyl-CoA. CaiB and CaiD were identified to be the two components of the crotonobetaine hydrating system, already described. CaiB and caiD were cloned and expressed in Escherichia coli. After purification of both proteins, their individual enzymatic functions were solved. CaiB acts as betainyl-CoA transferase specific for carnitine, crotonobetaine, gamma-butyrobetaine and its CoA derivatives. Transferase reaction proceeds, following a sequential bisubstrate mechanism. CaiD was identified to be a crotonobetainyl-CoA hydratase belonging to the crotononase superfamily. Because of amino acid sequence similarities, CaiC was suggested to be a betainyl-CoA ligase. Taken together, these results show that the metabolism of carnitine and crotonobetaine in Proteus sp. proceeds at the CoA level.

2-Enoyl-CoA hydratase 2 is the middle part of the mammalian peroxisomal multifunctional enzyme type 2 (MFE-2), which is known to be important in the beta-oxidation of very-long-chain and alpha-methyl-branched fatty acids as well as in the synthesis of bile acids. Here, we present the crystal structure of the hydratase 2 from the human MFE-2 to 3A resolution. The three-dimensional structure resembles the recently solved crystal structure of hydratase 2 from the yeast, Candida tropicalis, MFE-2 having a two-domain subunit structure with a C-domain complete hot-dog fold housing the active site, and an N-domain incomplete hot-dog fold housing the cavity for the aliphatic acyl part of the substrate molecule. The ability of human hydratase 2 to utilize such bulky compounds which are not physiological substrates for the fungal ortholog, e.g. CoA esters of C26 fatty acids, pristanic acid and di/trihydroxycholestanoic acids, is explained by a large hydrophobic cavity formed upon the movements of the extremely mobile loops I-III in the N-domain. In the unliganded form of human hydratase 2, however, the loop I blocks the entrance of fatty enoyl-CoAs with chain-length >C8. Therefore, we expect that upon binding of substrates bulkier than C8, the loop I gives way, contemporaneously causing a secondary effect in the CoA-binding pocket and/or active site required for efficient hydration reaction. This structural feature would explain the inactivity of human hydratase 2 towards short-chain substrates. The solved structure is also used as a tool for analyzing the various inactivating mutations, identified among others in MFE-2-deficient patients. Since hydratase 2 is the last functional unit of mammalian MFE-2 whose structure has been solved, the organization of the functional units in the biologically active full-length enzyme is also discussed.

Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJ(Ac)) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid beta-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJ(Ac) by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJ(Ac) revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C(8)) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C(12)) was examined by using the mutated PhaJ(Ac) as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1(Ps)). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C(8)) and 3-hydroxydecanoate (C(10)) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.

In yeast, the second and the third reaction of the fatty-acid beta-oxidation spiral are catalysed by peroxisomal multifunctional enzyme type 2 (Mfe2p/Fox2p). This protein has two (3R)-hydroxyacyl-CoA dehydrogenase domains and a C-terminal 2-enoyl-CoA hydratase 2 domain. Here, the purification, crystallization and X-ray diffraction analysis of the hydratase 2 domain [CtMfe2p(dh(a+b)Delta)] from Candida tropicalis Mfe2p is reported. CtMfe2p(dh(a+b)Delta) was overexpressed as an enzymatically active recombinant protein and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to space group C2, with unit-cell parameters a = 178.57, b = 60.46, c = 130.85 A, beta = 94.48 degrees. Selenomethionine-labelled protein was used for a multi-wavelength anomalous dispersion (MAD) experiment. A three-wavelength data set suitable for MAD phasing was collected to 2.25 A resolution using synchrotron radiation.

The biochemical and molecular properties of the beta-oxidation enzymes from algae have not been investigated yet. The present study provides such data for the phylogenetically old alga Euglena (Euglena gracilis). A novel multifunctional beta-oxidation complex was purified to homogeneity by ammonium sulfate precipitation, density gradient centrifugation, and ion-exchange chromatography. Monospecific antibodies used in immunocytochemical experiments revealed that the enzyme is located in mitochondria. The enzyme complex is composed of 3-hydroxyacyl-coenzyme A (-CoA) dehydrogenase, 2-enoyl-CoA hydratase, thiolase, and epimerase activities. The purified enzyme exhibits a native molecular mass of about 460 kD, consisting of 45.5-, 44.5-, 34-, and 32-kD subunits. Subunits dissociated from the complete complex revealed that the hydratase and the thiolase functions are located on the large subunits, whereas two dehydrogenase functions are located on the two smaller subunits. Epimerase activity was only measurable in the complete enzyme complex. From the use of stereoisomers and sequence data, it was concluded that the 2-enoyl-CoA hydratase catalyzes the formation of L-hydroxyacyl CoA isomers and that both of the different 3-hydroxyacyl-CoA dehydrogenase functions on the 32- and 34-kD subunits are specific to L-isomers as substrates, respectively. All of these data suggest that the Euglena enzyme belongs to the family of beta-oxidation enzymes that degrade acyl-CoAs via L-isomers and that it is composed of subunits comparable with subunits of monofunctional beta-oxidation enzymes. It is concluded that the Euglena enzyme phylogenetically developed from monospecific enzymes in archeons by non-covalent combination of subunits and presents an additional line for the evolutionary development of multifunctional beta-oxidation enzymes.

Cell free extracts of Galactomyces reessii contain a hydratase as the key enzyme for the transformation of 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid. Highest levels of hydratase activity were obtained during growth on isovaleric acid. The enzyme, an enoyl CoA hydratase, was purified 147-fold by precipitation with ammonium sulphate and successive chromatography over columns of DE-52, Blue Sepharose CL-6B and Sephacryl S-200. During purification, hydratase activity was measured spectrophotometrically (OD change at 263 nm) for 3-methylcrotonyl CoA and crotonyl CoA as substrates. The enzyme displayed highest activity with crotonyl CoA with a Kcat of 1,050,000 min(-1). The ratio of crotonyl CoA to 3-methylcrotonyl CoA activities was constant (20:1) during all steps of purification. The Kcat for crotonyl CoA was also about 20 times greater than the Kcat for 3-methylcrotonyl CoA (51,700 min(-1). The enzyme had pH and temperature optima at 7.0 and 35 degrees C, a native Mr of 260 +/- 4.5 kDa and a subunit Mr of 65 kDa, suggesting that the enzyme was a homotetramer. The pI of the purified hydratase was 5.5, and the N-terminal amino acid sequence was VPEGYAEDLLKGKMMRFFDS. Hydratase activity for 3-methylcrotonyl CoA was competitively inhibited by acetyl CoA, propionyl CoA and acetoacetyl CoA.

An (R)-trans-2,3-enoylacyl-CoA hydratase was purified to near-homogeneity from Rhodospirillum rubrum. Protein sequencing of enriched protein fractions allowed the construction of a degenerate oligonucleotide. The gene encoding the (R)-specific hydratase activity was cloned following three rounds of colony hybridization using the oligonucleotide, and overexpression of the gene in E. coli led to the purification of the enzyme to homogeneity. The purified enzyme used crotonyl-CoA, trans-2,3-pentenoyl-CoA, and trans-2,3-hexenoyl-CoA with approximately equal specificity as substrates in the hydration reaction. However, no activity was observed using trans-2,3-octenoyl-CoA as a substrate, but this compound did partially inhibit crotonyl-CoA hydration. Based on the nucleotide sequence, the protein has a monomeric molecular weight of 15.4 kDa and is a homotetramer in its native form as determined by gel filtration chromatography and native PAGE. The hydratase was expressed together with the PHA synthase from Thiocapsa pfennigii in E. coli strain DH5alpha. Growth of these strains on oleic acid resulted in the production of the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) .

We have identified the Saccharomyces cerevisiae gene ECI1 encoding Delta3-cis-Delta2-trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the beta-oxidation of (poly)unsaturated fatty acids. A mutant devoid of Eci1p was unable to grow on media containing unsaturated fatty acids such as oleic acid but was proficient for growth when a saturated fatty acid such as palmitic acid was the sole carbon source. Levels of ECI1 transcript were elevated in cells grown on oleic acid medium due to the presence in the ECI1 promoter of an oleate response element that bound the transcription factors Pip2p and Oaf1p. Eci1p was heterologously expressed in Escherichia coli and purified to homogeneity. It was found to be a hexameric protein with a subunit of molecular mass 32, 000 Da that converted 3-hexenoyl-CoA to trans-2-hexenoyl-CoA. Eci1p is the only known member of the hydratase/isomerase protein family with isomerase and/or 2-enoyl-CoA hydratase 1 activities that does not contain a conserved glutamate at its active site. Using a green fluorescent protein fusion, Eci1p was shown to be located in peroxisomes of wild-type yeast cells. Rat peroxisomal multifunctional enzyme type I containing Delta3-cis-Delta2-trans-enoyl-CoA isomerase activity was expressed in ECI1-deleted yeast cells, and this restored growth on oleic acid.

Many aromatic compounds can be metabolized by bacteria under anoxic conditions via benzoyl-CoA as the common intermediate. The central pathway of benzoyl-CoA metabolism is initiated by an ATP-driven reduction of the aromatic ring producing cyclohexa-1,5-diene-1-carbonyl-CoA. The 1,5-dienoyl-CoA intermediate is thought to be transformed to 6-hydroxycyclohex-1-ene-1-carbonyl-CoA by a specific dienoyl-CoA hydratase catalyzing the formal addition of water to one of the double bonds. This dienoyl-CoA hydratase was detected in the denitrifying bacterium Thauera aromatica after anaerobic growth with benzoate. Substrate and product were confirmed and a convenient spectrophotometric assay was developed. The equilibrium concentrations of substrate and product were almost equal. Enzyme activity was induced after anoxic growth with benzoate, in contrast to acetate. The enzyme of 28 kDa was purified from T. aromatica and was found to be highly specific for the cyclic 1,5-dienoyl-CoA. A second 29-kDa enoyl-CoA hydratase acted on crotonyl-CoA; this highly active enoyl-CoA hydratase also acted slowly on cyclohex-1-ene-1-carbonyl-CoA. The regulation of expression of dienoyl-CoA hydratase activity, the kinetic constants, the substrate specificity, and the specific activity of the enzyme in cell extract provide evidence that dienoyl-CoA hydratase is the second enzyme of the central benzoyl-CoA pathway of anaerobic aromatic metabolism in T. aromatica. Extracts of Rhodopseudomonas palustris contained high activity of cyclohex-1-ene-1-carbonyl-CoA hydratase, but no 1,5-dienoyl-CoA hydratase activity. It appears that a variant of the benzoyl-CoA pathway is operating in R. palustris in which hydration of the 1,5-dienoyl-CoA does not take place. Rather, cyclohex-1-ene-1-carbonyl-CoA is hydrated to 2-hydroxycyclohexane-1-carbonyl-CoA [corrected].

AU-rich elements within the 3' untranslated region of transcripts of lymphokines and some protooncogenes serve as signal for rapid mRNA degradation. By using an AUUUA matrix, we have affinity-purified a 32-kDa protein, microsequenced it, and cloned the corresponding cDNA. In vitro, the recombinant protein bound specifically to AU-rich transcripts, including those for interleukin 3, granulocyte/macrophage colony-stimulating factor, c-fos, and c-myc. Sequence analysis revealed an unexpected homology to enoyl-CoA hydratase (EC 4.2.1.17), and the recombinant protein showed a low degree of the enzymatic activity. Thus, this gene, designated AUH, encodes an RNA binding protein with intrinsic enzymatic activity. Protein immobilized on an AUUUA matrix was enzymatically active, suggesting that hydratase and AU-binding functions are located on distinct domains within a single polypeptide.

Mitochondrial 2-enoyl-CoA hydratase (mECH) and 3,2-trans-enoyl-CoA isomerase (mECI), two enzymes which catalyze totally different reactions in fatty acid beta-oxidation, belong to the low-similarity hydratase/isomerase enzyme superfamily. Their substrates and reaction mechanisms are similar [Müller-Newen, G. & Stoffel, W. (1993) Biochemistry 32, 11,405-11,412]. Glu164 of mECH is the only amino acid with a protic side chain that is conserved in these monofunctional and polyfunctional enzymes with 2-enoyl-CoA hydratase and 3,2-trans-enoyl-CoA isomerase activities. We tested our hypothesis that Glu164 of mECH is the putative active-site amino acid responsible for the base-catalyzed alpha-deprotonation in the hydratase/dehydrase and isomerase reaction. We functionally expressed rat liver mECH wild-type and [E164Q] mutant enzymes in Escherichia coli. Characterization of the purified wild-type and mutant enzymes revealed that the replacement of Glu164 by Gln lowers the kcat value more than 100,000-fold, whereas the Km value is only moderately affected. We have demonstrated in a previous study that Glu165 is indispensable for the 3,2-trans-enoyl-CoA isomerase activity. Taking these results together, we conclude that the conserved glutamic acid is the essential basic group in the active sites of 2-enoyl-CoA hydratase (Glu164) and 3,2-trans-enoyl-CoA isomerase (Glu165), and that these enzymes are not only evolutionarily but also functionally and mechanistically related.

The subunit locations of the component enzymes of the pig heart trifunctional mitochondrial beta-oxidation complex are suggested by analyzing the primary structure of the large subunit of this membrane-bound multienzyme complex [Yang S.-Y. et al. (1994) Biochem. biophys. Res. Commun. 198, 431-437] with those of the subunits of the E. coli fatty acid oxidation complex and the corresponding mitochondrial matrix beta-oxidation enzymes. Long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase are located in the amino-terminal and the central regions of the 79 kDa polypeptide, respectively, whereas the long-chain 3-ketoacyl-CoA thiolase is associated with the 46 kDa subunit of this complex. The pig heart mitochondrial bifunctional beta-oxidation enzyme is more homologous to the large subunit of the prokaryotic fatty acid oxidation complex than to the peroxisomal trifunctional beta-oxidation enzyme. The evolutionary trees of 3-hydroxyacyl-CoA dehydrogenases and enoyl-CoA hydratases suggest that the mitochondrial inner membrane-bound bifunctional beta-oxidation enzyme and the corresponding matrix monofunctional beta-oxidation enzymes are more remotely related to each other than to their corresponding prokaryotic enzymes, and that the genes of E. coli multifunctional fatty acid oxidation protein and pig heart mitochondrial bifunctional beta-oxidation enzyme diverged after the appearance of eukaryotic cells.

A simple and rapid purification procedure for hepatic peroxisomal multifunctional enzyme (delta 3, delta 2-enoyl-CoA isomerase/enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase) from clofibrate treated mice is described. The purification is achieved within two days using ion-exchange chromatography and an easily prepared affinity resin. The overall yield is 10% or more after a 100-fold enrichment from the cytosolic fraction of liver tissue. The native enzyme is a monomer with a molecular mass of 75 kDa. The protein is blocked in the N-terminus but internal amino acid sequences was obtained after proteolytic cleavage. Western blot analysis indicated proteolysis of multifunctional enzyme in different subcellular fractions derived from liver tissue. The hydratase activity of the enzyme is heat-labile and highly dependent on the concentration of Tris buffer or potassium chloride present. Optimal activity was found around 37 degrees C and pH 7. The enzyme also shows dehydrogenase and isomerase activity.

The effect of in vivo exposure to perfluoro-n-octanoic and perfluoro-n-decanoic acids was examined in the rat liver by two-dimensional electrophoresis (2DE). Using nonequilibrium pH-gradient electrophoresis in the first dimension separation, proteins associated with the mitochondrial/peroxisomal cell fraction were observed and immunologically identified. Conspicuous inductions in peroxisomal enoyl-CoA hydratase and other proteins of the peroxisomal beta-oxidative pathway were observed following single-dose exposure to each compound. The abundance of the tentatively-identified mitochondrial equivalent, crotonase, was not altered by these intoxications. These results confirm previous observations of perfluorocarboxylic acid toxicity and support the use of 2D protein-pattern alterations in biomarker research. The ability to identify this type of alteration via 2DE, in association with specific toxic effects by chemically related compounds, may provide new and additional markers for chemical-induced tissue damage.

Epiermization of 3-hydroxyacyl-CoA, which has been shown to occur as a two-step dehydration-hydration reaction (Hiltunen, J. K., Palosaari, P. M., and Kunau, W.-H. (1989) J. Biol. Chem. 264, 13536-13540; Smeland, E., Jianxun, L., Chu, C., Cuebas, D., and Schulz, H. (1989) Biochem. Biophys. Res. Commun. 160, 988-992) was studied in rat liver. Subcellular fractionations of rat liver on different density gradients revealed a dual distribution of activity, catalyzing dehydration of D-3-hydroxydecanoyl-CoA to trans-2-decenoyl-CoA (hydratase 2) in both peroxisomal and microsomal compartments. Both hydratase 2 activity peaks were separated by dye ligand chromatography from the extract of the combined heavy and light mitochondrial fractions. The activity eluted at low salt was identified as the microsomal isoform and was purified to apparent homogeneity. The M(r) of the native protein (subunit) was found to be 60,000 (31,500), indicating that it is homodimeric. The enzyme activity was inhibited by IgGs isolated from antisera raised against the denatured subunit. The activity eluted at high salt was tentatively identified to be peroxisomal of origin, and the M(r) of the native protein (subunit) was determined to be 62,000 (33,500). The peroxisomal enzyme was not recognized by the antibody to its microsomal counterpart. Analysis of the reaction products of microsomal enzyme activity by gas chromatography-mass spectrometry showed that the enzyme catalyzed reversibly hydration/dehydration between trans-2-enoyl-CoA and D-3-hydroxyacyl-CoA, but L-3-hydroxydecanoyl-CoA was not dehydrated to delta 2-enoyl-CoA compounds. Similar reaction characteristics were also determined for the peroxisomal hydratase by using stereospecific auxiliary enzymes. The present data demonstrate that rat liver contains microsomal and peroxisomal proteins possessing hydratase 2 activities. Although their kinetic properties are similar, immunological data, subunit sizes, and chromatographic evidence clearly indicate that they are different enzymes. Comparisons with other hydratases revealed that the microsomal and peroxisomal hydratase 2 described in the present work are proteins that have not been previously purified.

The peroxisome proliferator (PP) nafenopin (NAF) enhanced tumor development in rat liver through promotion of a subtype of putative preneoplastic cell foci, characterized by weak cytoplasmic basophilia. In order to elucidate the selective growth advantage of these weakly basophilic foci (WBF) we investigated the effects of NAF on their metabolic phenotype and DNA synthesis. In WBF, as well as in other foci subpopulations and in hepatocellular carcinomas the occurrence of five NAF-inducible enzymes, i.e. of peroxisomal beta-oxidation (acyl-CoA oxidase, bifunctional protein and thiolase), catalase and cytochrome P-452 was studied by immunohistochemical methods. In untreated livers almost all foci were stained with the same intensity as the surrounding tissue. When NAF was applied, most of the liver foci showed considerably less staining than the non-focal parenchyma in which pronounced enzyme induction had occurred. However, the subpopulation of WBF showed a more heterogeneous pattern of enzyme expression varying from less to even more than in the adjacent tissue. A similarly broad range of expression of peroxisomal enzymes was found in hepatocellular carcinomas. On average, however, the tumors exhibited less staining and lower activity of peroxisomal beta-oxidation than the surrounding parenchyma. WBF always showed higher rates of DNA synthesis than other foci subtypes and unaltered liver. In approximately one-third of these foci DNA synthesis was found to be enhanced concomitantly with elevated expression of peroxisomal beta-oxidation enzymes. In conclusion, WBF may have a selective growth advantage as they 'overrespond' to the inducing effects of NAF on DNA synthesis and peroxisomal enzymes.

TNF-alpha is a potent cytokine which induces marked hyperlipidemia. Because of the important role of peroxisomes in lipid metabolism we investigated the effects of human recombinant TNF-alpha upon rat liver peroxisomal enzymes. Sixteen hours after the administration of a single dose of 25 micrograms of TNF-alpha to male rats the activity of peroxisomal fatty acyl-CoA oxidase was reduced by 50%. This was confirmed also by immunoblotting and by quantitative immunoelectron microscopy which in addition revealed substantial reduction of the trifunctional protein (hydratase-dehydrogenase-isomerase) in peroxisomes. These observations suggest that the suppression of peroxisomal beta-oxidation may contribute to the perturbation of the isomerase) in peroxisomes. These observations suggest that the suppression of peroxisomal beta-oxidation may contribute to the perturbation of the lipid metabolism induced by TNF-alpha.

The present study provides strong evidence that the previously isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC 4.2.1.17), believed to be a component of the fatty acid chain-elongation system, is derived, not from the endoplasmic reticulum, but rather from the peroxisomes. The isolated dehydrase was purified over 3000-fold and showed optimal enzymic activity toward beta-hydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of 8-10. The purified preparation (VDH) displayed a pH optimum at 7.5 with beta-hydroxydecanoyl-CoA, and at 6.0 with beta-hydroxystearoyl-CoA. Competitive-inhibition studies suggested that VDH contained dehydrase isoforms, and SDS/PAGE showed three major bands at 47, 71 and 78 kDa, all of which reacted to antibody raised to the purified preparation. Immunocytochemical studies with anti-rabbit IgG to VDH unequivocally demonstrated gold particles randomly distributed throughout the peroxisomal matrix of liver sections from both untreated and di-(2-ethylhexyl) phthalate-treated rats. No labelling was associated with endoplasmic reticulum or with the microsomal fraction. Substrate-specificity studies and the use of antibodies to VDH and to the peroxisomal trifunctional protein indicated that VDH and the latter are separate enzymes. On the other hand, the VDH possesses biochemical characteristics similar to those of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265, 13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264, 13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoyl-CoA as substrates, but both enzymes convert trans-2-enoyl substrates into the D-isomer only. In addition, the VDH also contained beta-oxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase) activity, which co-purified with the dehydrase.

Long-chain 3-hydroxyacyl-CoA dehydrogenase was extracted from the washed membrane fraction of frozen rat liver mitochondria with buffer containing detergent and then was purified. This enzyme is an oligomer with a molecular mass of 460 kDa and consisted of 4 mol of large polypeptide (79 kDa) and 4 mol of small polypeptides (51 and 49 kDa). The purified enzyme preparation was concluded to be free from the following enzymes based on marked differences in behavior of the enzyme during purification, molecular masses of the native enzyme and subunits, and immunochemical properties: enoyl-CoA hydratase, short-chain 3-hydroxyacyl-CoA dehydrogenase, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases. The purified enzyme exhibited activities toward enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase together with the long-chain 3-hydroxyacyl-CoA dehydrogenase activity. The carbon chain length specificities of these three activities of this enzyme differed from those of the other enzymes. Therefore, it is concluded that this enzyme is not long-chain 3-hydroxyacyl-CoA dehydrogenase; rather, it is enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.

A trifunctional beta-oxidation protein, designated TFP, was purified to apparent homogeneity from oleate-induced mycelia of Neurospora crassa. 2-Enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase activities copurified in constant ratios with this protein when crude extracts were subjected to cation-exchange, dye-ligand, and adsorption chromatography. Trifunctionality was substantiated by coinciding enzyme activity ratios during the last two purification steps and additional chromatographic steps. The enzyme was shown to be a 365-kDa tetramer of subunits with a molecular mass of 93 kDa. Several lines of evidence suggest that these subunits are identical. Monospecific antibodies raised against the homogenous protein specifically precipitated the three enzymatic activities of TFP. Immunoblotting of fractions obtained after sucrose density gradient centrifugation of a crude extract indicated that TFP was exclusively localized in glyoxysome-like microbodies. The beta-oxidation system of N. crassa is structurally related to those of peroxisomes despite the presence of an acyl-CoA dehydrogenase rather than an acyl-CoA oxidase. A mitochondrial 2-enoyl-CoA hydratase activity was separated from TFP and purified to apparent homogeneity. The absence of all other beta-oxidation activities from mitochondria suggests that this organelle and its 2-enoyl-CoA hydratase are not involved in fatty acid degradation in N. crassa.

Determining the sequence of bond cleavages, and consequently the nature of intermediates, in enzyme-catalyzed reactions is a major goal of mechanistic enzymology. When significant primary isotope effects on V/K are observed for two different bond cleavages, both bonds may be broken in the same transition state or they can reflect two different transition states that are of nearly identical energy and consequently both are partially rate limiting. For the crotonase-catalyzed dehydration of 3-hydroxybutyrylpantetheine, the primary D(V/K) and 18(V/K) are 1.60 and 1.053 [Bahnson, B. J., & Anderson, V. E. (1989) Biochemistry 28, 4173-4181], respectively. In this case, double isotope effects can discriminate between the two possibilities [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114; Belasco, J. G., Albery, W. J., & Knowles, J. R. (1983) J. Am. Chem. Soc. 105, 2475-2477]. The ratio of the alpha-secondary D(V/K) for the hydration of crotonylpantetheine catalyzed by crotonase in H2O and D2O has been determined to be 1.003 +/- 0.006. The invariance of the alpha-secondary effect where the chemical reaction is completely rate determining requires that both bond cleavages be concerted or that the substitution of 2H at the primary position not significantly alter the partitioning of a hypothetical carbanion. The observation of a solvent discrimination isotope effect determined from the relative incorporation of 2H from 50% D2O of 1.60 +/- 0.03, identical with the primary D(V/K), and the determination that the rate of exchange of the abstracted proton with solvent proceeds at less than 3% of the overall reaction rate also fail to provide evidence for a carbanion intermediate and are consistent with a concerted reaction. Identical primary D(V/K)s determined in H2O and D2O indicate that there is not a significant solvent isotope effect on C-O bond cleavage. The isotope ratios determined in these studies were performed by negative ion chemical ionization whole molecule mass spectrometry of the pentafluorobenzyl esters, a new method whose validity is established by comparison with previously determined kinetic and equilibrium isotope effects.

Peroxisomal delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) was studied in the liver of rats treated with clofibrate. The mitochondrial and peroxisomal isoenzymes were separated chromatographically and the peroxisomal isomerase purified to apparent homogeneity. In addition to the isomerization of 3-enoyl-CoA esters, the purified protein also catalyzed hydration of trans-2-enoyl-CoA and oxidation of L-3-hydroxyacyl-CoA. Incubation of the purified protein with trans-3-decenoyl-CoA, NAD+, and Mg2+ resulted in an increase in absorbance at 303 nm, indicating the formation of 3-ketoacyl-CoA. The protein purified was monomeric, with an estimated molecular weight of 78,000. In immunoblotting it was recognized by the antibody to peroxisomal bifunctional protein from rat liver. Comparison of the amino acid sequences of cyanogen bromide cleaved isomerase with the known sequence of the peroxisomal bifunctional protein from the rat identified them as the same molecule. In control experiments, the peroxisomal bifunctional protein purified according to published methods also catalyzed delta 3, delta 2-enoyl-CoA isomerization. This means that the bifunctional protein of rat liver is in fact a trifunctional enzyme possessing delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase (EC 4.2.1.17), and L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities in the same polypeptide.

Acetoacetyl coenzyme A (acetoacetyl-CoA) thiolase, an enzyme required for short-chain fatty acid degradation, has been purified to near homogeneity from Caulobacter crescentus. The relative heat stability of this enzyme allowed it to be separated from beta-ketoacyl-CoA thiolase. The purification scheme minus the heating step also permitted the copurification of crotonase and 3-hydroxyacyl-CoA dehydrogenase. These activities are in a multienzyme complex in Escherichia coli, but a similar complex was not observed in C. crescentus. Instead, separate proteins differing in enzymatic activity were detected, analogous to the beta-oxidation enzymes that have been isolated from Clostridium acetobutylicum and from mitochondria of higher eucaryotes. In these cells, as appears to be the case with C. crescentus, the individual enzymes form multimers of identical subunits.

2-Enoyl-CoA reductase was purified 150-fold from the crude extract of Mycobacterium smegmatis. The purified reductase required NADH, but not NADPH, as a reductant and catalyzed the reduction of C4 to C16 enoyl-CoAs, though the activities toward shorter chain substrates (c4 and C6) were very low. Thiolase was also partially purified from the same source. These two enzymes were used, together with 3-hydroxyacyl-CoA dehydrogenase and the two forms of enoyl-CoA hydratase (hydratases I and II) previously purified from the same source, to reconstitute fatty acid elongation activity. The products formed from [1-14C]acetyl-CoA and decanoyl-CoA in this reconstituted system were analyzed by thin-layer chromatography and radio-gas-liquid chromatography. The system containing hydratase II produced laurate and 3-hydroxylaurate (in the form of their CoA esters) and the ratio of laurate to 3-hydroxylaurate increased as the incubation time was increased. The system containing hydratase I produced only 3-hydroxylaurate. 3-Hydroxylaurate was also the only product when enoyl-CoA reductase was omitted from the system containing hydratase II. It is concluded that hydratase II, but not hydratase I, is functional in fatty acid elongation by M. smegmatis and that enoyl-CoA reductase is also essential for the reaction. CoA and NAD+ inhibited the reconstituted elongation activity in competition with acetyl-CoA and NADH, respectively.

Two forms of enoyl-CoA hydratase (hydratases I and II), which are different from each other in substrate specificity, were found in a crude extract of Mycobacterium smegmatis. Hydratase I was more active with crotonyl-CoA as a substrate than with decenoyl-CoA, whereas the reverse was the case for hydratase II. Hydratase I was purified 688-fold to homogeneity with a yield of 14.5% from the crude extract. Its molecular weight was estimated to be 16,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 30,000 by gel filtration, suggesting that the enzyme is dimeric. Hydratase II was also partially purified. The Vmax of hydratase I decreased progressively with increase in the carbon-chain length of the substrate from 2,488 units/mg for crotonyl-CoA to 154 units/mg for hexadecenoyl-CoA, whereas the Km values for crotonyl-CoA (82 microM), decenoyl-CoA (91 microM), and hexadecenoyl-CoA (105 microM) were similar. Both hydratases were inhibited by acetoacetyl-CoA and pCMS, but not by N-ethylmaleimide or monoiodoacetate.

Glyoxysomal enzymes, being necessary during seed germination, are already synthesized at the stage of seed maturation. Two stages of embryogenesis of cucumber seeds (Cucumis sativus) were investigated. One was characterized by the presence of microbodies showing catalase and enoyl-CoA hydratase activities. Microbodies at a later stage contained, in addition, malate synthase and isocitrate lyase. The biosynthesis of three microbody components was followed in a pulse chase-labelling experiment which demonstrated that the biosynthesis of cytosolic species of malate synthase, isocitrate lyase and enoyl-CoA hydratase preceded the appearance of these proteins in microbodies.

A molecular understanding of genetic disease in which peroxisomal functions are impaired depends on analysis of the structure of normal and mutant enzymes of peroxisomes. We report experiments describing the isolation, characterization, and immunocytochemical localization of enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (PBE) of the peroxisomal fatty acid beta-oxidation system from normal human liver and compared it with that of rat liver enzyme. The human enzyme, purified approximately equal to 2300-fold by ion-exchange chromatography, is homogeneous as judged by NaDodSO4/PAGE. This PBE is localized exclusively in the matrix of peroxisomes in liver cells by the protein A/gold immunocytochemical method. The human PBE is similar to rat enzyme in size (Mr, approximately equal to 79,000), isoelectric point (pI, 9.8), pH optima, molecular structure as observed by rotary shadowing, and peptide pattern on NaDodSO4/PAGE after proteolytic digestion with Staphylococcus aureus V8 protease. The human and rat enzymes differed in their immunological properties by having partial identity with each other; this is reflected in their slightly dissimilar composition of the amino acids aspartic acid, threonine, glutamic acid, tyrosine, and glycine. COOH-terminal amino acid were similar for both the enzymes: -Gly-Ser-Leu-Ile-COOH. These results suggest that the human and rat liver PBE may be different in their amino acid sequences at their antigenic sites.

The purpose of the present study was to determine if the enoyl coenzyme A hydratase formed by Pseudomonas putida during growth on isoleucine was a unique enzyme specific for isoleucine metabolism. The highest levels of the hydratase were formed during growth on isoleucine intermediates and the lowest levels during growth on glutamate and glucose. Data from growth experiments revealed that 2-methyl-3-hydroxybutyryl coenzyme A hydratase, an enzyme unique to isoleucine metabolism and enoyl coenzyme A hydratase were coordinately induced, but that 3-hydroxyacyl coenzyme A dehydrogenase was under separate control. The hydratase was purified 180-fold from isoleucine cells, and its physical and catalytic properties reported. The highest activity was with crotonyl coenzyme A,Vmax = 1100 x 10(3) moles/min mole enzyme, next was tiglyl coenzyme A, Vmax = 61 x 10(3) moles/min mole enzyme, and last was 3-methyl-crotonyl coenzyme A, Vmax = 2.3 x 10(3) moles/min mole enzyme. Enzyme purified from butyrate cells had the same elution patterns during column chromatography and catalytic properties as the enzyme from isoleucine cells. These data support the conclusion that a single enzyme in P. putida is responsible for the hydration of both tiglyl coenzyme A and crotonyl coenzyme A.

An enzyme system of Mycobacterium smegmatis catalyzing the elongation of medium-chain fatty acids with acetyl-CoA was obtained free from de novo fatty acid synthetase by ammonium sulfate fractionation. The system was resolved by gel filtration and DEAE-cellulose chromatography into three fractions, all of which were required for reconstitution of the elongation activity. The three fractions were highly purified enoyl-CoA hydratase, highly purified 3-hydroxyacyl-CoA dehydrogenase, and a fraction containing both enoyl-CoA reductase and thiolase. The reconstituted system was avidin-insenstive, required NADH as a sole hydrogen donor, and was sensitive to pCMB, but not to N-ethylmaleimide or monoiodoacetate. Decanoyl-CoA and octanoyl-CoA were the best primers for the elongation system. When decanoyl-CoA was used as the primer, the major product was found to be a lauroyl derivative (probably lauroyl-CoA). Evidence was obtained suggesting that acyl-CoA dehydrogenase, catalyzing the first step of beta-oxidation, was not functional in the elongation system.

The solubilization and partial purification of beta-hydroxyacyl-CoA dehydrase from rat liver microsomes has been accomplished through deoxycholate solubilization, ammonium sulfate fractionation, and ion exchange chromatography. A purification of about 90-fold based on total soluble activity was achieved, with an overall yield of 40%. However, the initial solubilization is accompanied by the loss of the secondary portion of the v/s curve observed with intact microsomes. The enzyme requires detergent during the purification procedure to remain "soluble," and is strongly activated by the inclusion of Triton-X-100 at concentrations above its critical micelle concentration in the assay mixture. In addition a preference for micelles has been inferred based on discontinuities in the v/s curves relative to the measured critical micelle concentration of the substrates in the absence of Triton X-100. Kinetic parameters calculated on the basis of micelle-specific activity indicated that beta-hydroxyacyl-CoA substrates possessing even-numbered alkyl chains from 14 to 20 carbon atoms differed little in Vm', but had progressively larger Km' as the chain length increased. The partially purified preparation was also active with beta-hydroxy-8,11-eicosadienoyl-CoA; and with 2-trans-enoyl-CoA substrates in a reverse (hydration) reaction.

Mitochondrial and peroxisomal enoyl-CoA hydratases were purified from rat liver. The mitochondrial enzyme, with a molecular weight of 161,000, was composed of 6 identical subunits. The molecular structure of the rat liver enzyme was very similar to that of the bovine liver enzyme. Acetoacetyl-CoA was a competitive inhibitor of the mitochondrial enzymes. The results of titration of the rat liver enzyme with acetoacetyl-CoA suggest that 3 subunits of the enzyme exhibit catalytic activity. The catalytic properties of the enzyme were studied. The peroxisomal enzyme was composed of one polypeptide with a molecular weight of 70,000-81,000. Some of the enzyme molecules were shown to be cleaved to two polypeptides in the cell by the following methods: amino acid analysis, peptide mapping and immunoprecipitin reaction. The catalytic properties of the peroxisomal enzyme were different from those of the mitochondrial enzyme. The peroxisomal enzyme is a bifunctional enzyme exhibiting 3-hydroxyacyl-CoA dehydrogenase activity. Studies on the titration with acetoacetyl-CoA, the effects of salts, SH titration and proteolytic inactivation suggest that the active centers for these two reactions are located at different sites.

Controlled proteolytic cleavage of pigeon liver fatty acid synthetase with elastase (4% w/w) for 5 h yields two peptides that are designated II and IV. After 5 h of proteolysis the incubation mixture containing these peptides retains all of the component enzyme activities of the fatty acid synthetase complex. The two peptides are then separated by chromatography on an Affi-Gel Blue column. Gel filtration of the fraction containing peptide II yields a homogeneous peptide as shown by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of this peptide has been estimated to be 130 000 by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, size exclusion chromatography, and amino acid analysis. The sedimentation coefficient for peptide II is approximately 7.4S. Peptide II contains the domains for the beta-ketoacyl and enoyl reductases and beta-hydroxyacyl dehydrase activities of the fatty acid synthetase complex.

1. Enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase copurified when extracts from cotyledons of 5-day-old cucumber seedlings were fractionated by acetone precipitation, ion-exchange chromatography on CM-cellulose and affinity chromatography on blue-dextran-Sepharose. The protein was purified 600-fold with 16% recovery. 2. Bifunctionality of the protein was substantiated by exactly coinciding activity profiles upon chromatography on hydroxyapatite, CM-cellulose, or blue-dextran-Sepharose respectively. Analysis of the purified protein using isoelectric focusing as well as native electrophoresis confirmed the presence of a bifunctional protein. A monospecific antiserum could be raised against the homogeneous protein. 3. Further characterization of the protein revealed that the two enzyme activities are contained in a single peptide chain of Mr 75000. An extremely alkaline isoelectric point of pH 9.8 was determined. 4. The bifunctional protein was localized in glyoxysomes which were shown to be the exclusive site of beta-oxidation at this stage of germination. At least 10% of the enzyme were attributable to the organelle's membrane.

The isolation of three proteins in crystalline form from ground beef liver is described. These proteins are FTBL protein (Arch. Biochem. Biophys. 188, 251-265 (1978), crotonase, and catalase. Crotonase is isolated by crystallization from a 32% acetone extract of the ground liver. FTBL protein and catalase can subsequently be isolated from the same extract. For optimal yield and ease of isolation, FTBL protein is isolated from a 46.5% acetone extract from which catalase can subsequently be crystallized by dialysis. The isolation of FTBL protein as well as the isolation of catalase involves a preliminary fractional precipitation and solution before crystallization can be achieved. Isopropanol can be substituted for acetone in the isolation of the above three proteins and in the case of catalase, results in an exceptionally high yield. Methods for the recrystallization of the proteins are presented and the role of organic solvents in recrystallization is discussed.

Interconversion of D- and L-isomers of 3-hydroxy-decanoyl-CoA was catalyzed by rat liver homogenate. Cation exchange chromatography followed by ammonium sulfate precipitation and PBE-94 chromatofocusing column was used to separate the peroxisomal bifunctional protein, the classic 2-enoyl-CoA hydratase (crotonase), and a novel 2-enoyl-CoA hydratase. Epimerization activity was lost during the last purification step. None of the above proteins was capable of catalyzing the epimerization by itself, but reconstitution was achieved by recombining crotonase and the novel 2-enoyl-CoA hydratase. Since hydration by the latter enzyme follows a different stereochemical course from that with crotonase, these two hydratases are distinguished as 2-enoyl-CoA hydratase 1 (crotonase) and 2-enoyl-CoA hydratase 2 (the novel hydratase). The data strongly suggested that epimerization in the rat liver proceeds via dehydration-hydration catalyzed by the two different hydratases. The intermediate of this two step mechanism appears to be trans-2-enoyl-CoA.

The ability of 0.4 M KCl to extract over 80% of a short-chain beta-hydroxyacyl-CoA dehydrase from rat hepatic endoplasmic reticulum, while more than 80% of the long-chain beta-hydroxyacyl-CoA dehydrase component of the fatty acid chain elongation system remains intact, confirms the existence of more than one hepatic microsomal dehydrase. Following extraction from the microsomal membrane, the short-chain dehydrase undergoes, at least, a two-fold activation. Employing even-numbered trans-2-enoyl-CoA substrates ranging in carbon chain length from 4 to 16, the highest dehydrase specific activity of 16 mumol min-1 mg protein-1 was obtained with trans-2-hexenoyl-CoA; crotonyl-CoA was the second most active substrate, followed by 8 greater than 10 greater than 12 greater than 14 greater than 16. The specific activity of the short-chain dehydrase with trans-2-hexadecenoyl-CoA (C-16) was only 3% of that observed with the trans-2-hexenoyl-CoA. With crotonyl-CoA or beta-hydroxybutyryl-CoA as substrates, HPLC was employed to identify the products, beta-hydroxybutyryl-CoA, of the hydration reaction, or crotonyl-CoA, of the reverse dehydration reaction. It was also observed that the short-chain dehydrase catalyzed the formation of both D(-) and L(+) stereoisomers of beta-hydroxybutyryl-CoA. The equilibrium constant for the dehydrase-catalyzed reaction determined at pH 7.4 and 35 degrees C, was calculated to be 6.38 X 10(-2) M-1, while the standard free energy change was -775 cal/mol, results similar to those obtained with crystalline crotonase. Finally, based on membrane fraction marker enzymes, substrate specificity, and heat lability of the dehydrase, it was concluded that the microsomal membrane contains a short-chain beta-hydroxyacyl-CoA dehydrase which is separate from the mitochondrial crotonase.

The enzymatic apparatus of fatty acid beta-oxidation in peroxisomes and glyoxysomes includes a multifunctional protein. Two forms of this protein were detected in extracts from cotyledons of germinating cucumber seeds and separated on hydroxylapatite. The two proteins purified to apparent homogeneity possessed enoyl-CoA hydratase, 3-hydroxyacyl-CoA epimerase, and 3-hydroxyacyl-CoA dehydrogenase activity; the proteins are therefore trifunctional. Analysis of molecular structures and kinetic parameters of the two enzyme forms revealed significant differences in size and amino acid composition. The two proteins were characterized as monomers exhibiting molecular weights of 74,000 and 76,500. Likewise, the data obtained with limited proteolysis proved the occurrence of two independent proteins. Immunological comparisons were performed with antibodies raised against the 76.5-kDa protein. They indicated a weak relationship between the two proteins. From that we conclude that within one type of organelle, i.e., glyoxysome, two isoenzymes with multiple functions are located.