Substrate specificities of peroxisomal members of short-chain alcohol dehydrogenase superfamily: expression and characterization of dehydrogenase part of Candida tropicalis multifunctional enzyme

In addition to several other enzymes, the short-chain alcohol dehydrogenase superfamily includes a group of peroxisomal multifunctional enzymes involved in fatty acid and cholesterol side-chain beta-oxidation. Mammalian peroxisomal multifunctional enzyme type 2 (perMFE-2) is a 2-enoyl-CoA hydratase-2/(R)-3-hydroxyacyl-CoA dehydrogenase. As has been shown previously, perMFE-2 hydrates (24E)-3alpha,7alpha, 12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA to (24R, 25R)-3alpha, 7alpha,12alpha,24xi-tetrahydroxy-5beta-choles tanoyl-CoA, which has been characterized as a physiological intermediate in cholic acid synthesis. Out of four possible stereoisomers of 3alpha,7alpha, 12alpha,24xi-tetrahydroxy-5beta-cholestanoyl-CoA , the mammalian perMFE-2 dehydrogenates only the (24R,25R)-isomer. The yeast peroxisomal multifunctional enzyme (MFE) was first described as 2-enoyl-CoA hydratase-2/(R)-3-hydroxyacyl-CoA dehydrogenase. To investigate the stereospecificity of yeast peroxisomal MFE, the two dehydrogenase domains of C. tropicalis MFE were expressed in E. coli as a 65 kDa recombinant protein. This protein catalyzes the dehydrogenation of straight-chain (R)-3-hydroxyacyl-CoAs, but it is devoid of (S)-3-hydroxyacyl-CoA dehydrogenase and 2-enoyl-CoA hydratase activities. The protein dehydrogenates (24R,25R)- and (24R, 25S)-isomers of 3alpha,7alpha, 12alpha, 24xi-tetrahydroxy-5beta-cholestanoyl-CoA. Interestingly, the protein also shows 17beta-estradiol dehydrogenase activity.As a monofunctional (R)-specific 3-hydroxyacyl-CoA dehydrogenase is currently unavailable, this recombinant enzyme can be used to study the stereochemistry of bile acid synthesis.     

A two-domain structure of one subunit explains unique features of eukaryotic hydratase 2

2-Enoyl-CoA hydratase 2, a part from multifunctional enzyme type 2, hydrates trans-2-enoyl-CoA to 3-hydroxyacyl-CoA in the (3R)-hydroxy-dependent route of peroxisomal beta-oxidation of fatty acids. Unliganded and (3R)-hydroxydecanoyl coenzyme A-complexed crystal structures of 2-enoyl-CoA hydratase 2 from Candida tropicalis multifunctional enzyme type 2 were solved to 1.95- and 2.35-A resolution, respectively. 2-Enoyl-CoA hydratase 2 is a dimeric, alpha+beta protein with a novel quaternary structure. The overall structure of the two-domain subunit of eukaryotic 2-enoyl-CoA hydratase 2 resembles the homodimeric, hot dog fold structures of prokaryotic (R)-specific 2-enoyl-CoA hydratase and beta-hydroxydecanoyl thiol ester dehydrase. Importantly, though, the eukaryotic hydratase 2 has a complete hot dog fold only in its C-domain, whereas the N-domain lacks a long central alpha-helix, thus creating space for bulkier substrates in the binding pocket and explaining the observed difference in substrate preference between eukaryotic and prokaryotic enzymes. Although the N- and C-domains have an identity of <10% at the amino acid level, they share a 50% identity at the nucleotide level and fold similarly. We suggest that a subunit of 2-enoyl-CoA hydratase 2 has evolved via a gene duplication with the concomitant loss of one catalytic site. The hydrogen bonding network of the active site of 2-enoyl-CoA hydratase 2 resembles the active site geometry of mitochondrial (S)-specific 2-enoyl-CoA hydratase 1, although in a mirror image fashion. This arrangement allows the reaction to occur by similar mechanism, supported by mutagenesis and mechanistic studies, although via reciprocal stereochemistry.     

Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1-¹⁴C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-¹⁴C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-¹⁴C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-¹⁴C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-¹⁴C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-¹⁴C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-¹⁴C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg⁻¹ h⁻¹, which is the highest ever reported for a plant system.     

Acyl-CoA elongase from a higher plant (Lunaria annua): metabolic intermediates of very-long-chain acyl-CoA products and substrate specificity

A particulate fraction (15,000 x g pellet) from developing seeds of honesty (Lunaria annua) was found to synthesize very-long-chain acyl-CoA thioesters in a manner similar to mammalian systems, i.e., via condensation of an acyl-CoA with malonyl-CoA yielding beta-ketoacyl-CoA, which is reduced to beta-hydroxyacyl-CoA, the latter dehydrated to trans-2-enoyl-CoA that is finally reduced to very-long-chain acyl-CoA. Reduced pyridine nucleotides (NADH/NADPH) are required for the reduction steps. In the absence of reduced pyridine nucleotides only the condensation reaction occurs. The acyl-CoA elongase does not exhibit any pronounced specificity for any of the saturated (14:0 to 20:0) or (n - 9)cis-monounsaturated (14:1 to 22:1) acyl-CoA substrates, although both the saturated and monounsaturated acyl-CoA substrates having chain lengths of C18 and C20 are elongated somewhat faster.     

Epimerization of 3-hydroxy-4-trans-decenoyl coenzyme A by a dehydration/hydration mechanism catalyzed by the multienzyme complex of fatty acid oxidation from Escherichia coli.

The mechanism of 3-hydroxyacyl-CoA epimerase (EC 5.1.2.3), which is associated with the multienzyme complex of fatty acid oxidation from Escherichia coli, was studied with D-3-hydroxy-4-trans-decenoyl-CoA as a substrate. The E. coli complex catalyzes the rapid and direct dehydration of D-3-hydroxy-4-trans-decenoyl-CoA to 2-trans,4-trans-decadienoyl-CoA, which is slowly hydrated to L-3-hydroxy-4-trans-decenoyl-CoA. A kinetic analysis of the epimerase and its partial reactions established that epimerization of 3-hydroxyacyl-CoAs occurs solely by a dehydration/hydration mechanism. The results of a substrate competition study with L-3-hydroxy-4-trans-decenoyl-CoA and its D-isomer, together with the conclusion from a sequence analysis of the large subunit of the E. coli complex (Yang, X.-Y., Schulz, H., Elzinga, M., and Yang, S.-Y. (1991) Biochemistry 30, 6788-6795), prompt the suggestion that a single active site is responsible for the dehydration of the D- and L-isomers of 3-hydroxyacyl-CoAs.     

The Endoplasmic reticulum-associated maize GL8 protein is a component of the acyl-coenzyme A elongase ivolved in the production of cuticular waxes

The gl8 gene is required for the normal accumulation of cuticular waxes on maize (Zea mays) seedling leaves. The predicted GL8 protein exhibits significant sequence similarity to a class of enzymes that catalyze the reduction of a ketone group to a hydroxyl group. Polyclonal antibodies raised against the recombinant Escherichia coli-expressed GL8 protein were used to investigate the function of this protein in planta. Subcellular fractionation experiments indicate that the GL8 protein is associated with the endoplasmic reticulum membranes. Furthermore, polyclonal antibodies raised against the partially purified leek (Allium porrum) microsomal acyl-coenzyme A (CoA) elongase can react with the E. coli-expressed GL8 protein. In addition, anti-GL8 immunoglobulin G inhibited the in vitro elongation of stearoyl-CoA by leek and maize microsomal acyl-CoA elongase. In combination, these findings indicate that the GL8 protein is a component of the acyl-CoA elongase. In addition, the finding that anti-GL8 immunoglobulin G did not significantly inhibit the 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA dehydrase, and (E) 2,3-enoyl-CoA reductase partial reactions of leek or maize acyl-CoA elongase lends further support to our previous hypothesis that the GL8 protein functions as a beta-ketoacyl reductase during the elongation of very long-chain fatty acids required for the production of cuticular waxes.     

Evidence for a peroxisomal fatty acid beta-oxidation involving D-3-hydroxyacyl-CoAs. Characterization of two forms of hydro-lyase that convert D-(-)-3-hydroxyacyl-CoA into 2-trans-enoyl-CoA

A novel D-(-)-3-hydroxyacyl-CoA hydro-lyase, forming 2-trans-enoyl-CoA and formerly designated as epimerase (EC 5.1.2.3), was extracted from fat-degrading cotyledons of cucumber seedlings. The enzyme, called D-3-hydroxyacyl-CoA hydro-lyase or D-specific 2-trans-enoyl-CoA hydratase, is shown to be required for the degradation of unsaturated fatty acids that contain double bonds extending from even-numbered C atoms. The D-3-hydroxyacyl-CoA hydro-lyase was exclusively localized within peroxisomes. A 10,000-fold purification by chromatography on a hydrophobic matrix, a cation exchanger, on hydroxyapatite and Mono S led to two proteins of apparent homogeneity, both exhibiting Mr of 65,000. The D-3-hydroxyacyl-CoA hydro-lyases are homodimers with slightly differing isoelectric points around pH = 9.0. They catalyze the conversion of 2-trans-enoyl-CoA into D-3-hydroxyacyl-CoA. The reverse reaction was observed but no reaction with 2-cis-enoyl-CoAs or L-3-hydroxyacyl-CoAs. 2-trans-Decenoyl-CoA was converted 10-times faster than 2-trans-butenoyl-CoA. The conversion of 4-cis-decenoyl-CoA into octenoyl-CoA was demonstrated in vitro with purified proteins with an assay mixture containing acyl-CoA oxidase, multifunctional protein, thiolase and the D-3-hydroxyacyl-CoA hydro-lyase. Comparisons of enzyme activities present in the cotyledons or isolated peroxisomes clearly show that the pathway via dienoyl-CoA reductase is much less effective than the sequence involving D-3-hydroxyacyl-CoA hydro-lyase.     

Inactivation of medium-chain acyl-CoA dehydrogenase by oct-4-en-2-ynoyl-CoA

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta-oxidation of fatty acids, which catalyzes the FAD-dependent oxidation of a variety of acyl-CoA substrates to the corresponding trans-2-enoyl-CoA thioesters. Oct-4-en-2-ynoyl-CoA was identified as a new irreversible inhibitor of acyl-CoA dehydrogenase, and kinetic parameters K(I) and k(inact) were determined to be 11 microM and 0.025 min(-1), respectively. Triple bond between C2 and C3 of the inhibitor was identified as the functional group responsible for enzyme inactivation, and Michael addition is proposed as the mechanism for this inactivation, which is a new pathway for inactivation of MCAD by inhibitors. The inhibitor may become a lead for further development for treating non-insulin-dependent diabetes mellitus.     

Identification of two mammalian reductases involved in the two-carbon fatty acyl elongation cascade

The de novo synthesis of fatty acids occurs in two distinct cellular compartments. Palmitate (16:0) is synthesized from acetyl-CoA and malonyl-CoA in the cytoplasm by the enzymes acetyl-CoA carboxylase 1 and fatty acid synthase. The synthesis of fatty acids longer than 16 carbons takes place in microsomes and utilizes malonyl-CoA as the carbon source. Each two-carbon addition requires four sequential reactions: condensation, reduction, dehydration, and a final reduction to form the elongated fatty acyl-CoA. The initial condensation reaction is the regulated and rate-controlling step in microsomal fatty acyl elongation. We previously reported the cDNA cloning and characterization of a murine long chain fatty acyl elongase (LCE) . Overexpression of LCE in cells resulted in the enhanced addition of two-carbon units to C12-C16 fatty acids, and evidence was provided that LCE catalyzed the initial condensation reaction of long chain fatty acid elongation. The remaining three enzymes in the elongation reaction have not been identified in mammals. Here, we report the identification and characterization of two mammalian enzymes that catalyze the 3-ketoacyl-CoA and trans-2,3-enoyl-CoA reduction reactions in long and very long chain fatty acid elongation, respectively.     

Induction of enzymes involved in fatty acid beta-oxidation in Pseudomonas fragi B-0771 cells grown in media supplemented with fatty acid.

Induction of the enzymes involved in fatty acid beta-oxidation in Pseudomonas fragi B-0771 cells grown in a medium containing straight chain saturated fatty acids was studied. The acyl-CoA dehydrogenase (ACDH) activity was induced during the exponential phase in cells grown in palmitic acid-supplemented medium, reached a maximum at the early stationary phase, and then gradually decreased thereafter. Changes in the overall activities of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase, both existing on the multienzyme complex (HDT) involved in fatty acid beta-oxidation, were similar to that in ACDH activity. Straight chain saturated fatty acids having more than 6 carbon atoms could induce both the ACDH and HDT activities, and C13-C15 fatty acids caused the greatest induction of both activities. Changes in the overall activities of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase correlated with that in the amount of the alpha-subunit of HDT during the entire culture period in the medium containing palmitic acid. Surprisingly, the stoichiometry of the alpha- and beta-subunit proteins of HDT was not maintained into the stationary phase culture, though the genes encoding the alpha- and beta-subunits are tandemly coded in bacterial genomic DNA.     

Hepatic microsomal short-chain beta-hydroxyacyl-CoA dehydrase distinct from the fatty acid elongation component: substrate specificity of the membrane-extracted enzyme

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.     

Mitochondrial trans-2-enoyl-CoA reductase of wax ester fermentation from Euglena gracilis defines a new family of enzymes involved in lipid synthesis

Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. An important enzyme of this unusual pathway is trans-2-enoyl-CoA reductase (EC 1.3.1.44), which catalyzes reduction of enoyl-CoA to acyl-CoA. Trans-2-enoyl-CoA reductase from Euglena was purified 1700-fold to electrophoretic homogeneity and was active with NADH and NADPH as the electron donor. The active enzyme is a monomer with molecular mass of 44 kDa. The amino acid sequence of tryptic peptides determined by electrospray ionization mass spectrometry were used to clone the corresponding cDNA, which encoded a polypeptide that, when expressed in Escherichia coli and purified by affinity chromatography, possessed trans-2-enoyl-CoA reductase activity close to that of the enzyme purified from Euglena. Trans-2-enoyl-CoA reductase activity is present in mitochondria and the mRNA is expressed under aerobic and anaerobic conditions. Using NADH, the recombinant enzyme accepted crotonyl-CoA (km=68 microm) and trans-2-hexenoyl-CoA (km=91 microm). In the crotonyl-CoA-dependent reaction, both NADH (km=109 microm) or NADPH (km=119 microm) were accepted, with 2-3-fold higher specific activities for NADH relative to NADPH. Trans-2-enoyl-CoA reductase homologues were not found among other eukaryotes, but are present as hypothetical reading frames of unknown function in sequenced genomes of many proteobacteria and a few Gram-positive eubacteria, where they occasionally occur next to genes involved in fatty acid and polyketide biosynthesis. Trans-2-enoyl-CoA reductase assigns a biochemical activity, NAD(P)H-dependent acyl-CoA synthesis from enoyl-CoA, to one member of this gene family of previously unknown function.     

Measurement of the acyl-CoA intermediates of beta-oxidation by h.p.l.c. with on-line radiochemical and photodiode-array detection. Application to the study of [U-14C]hexadecanoate oxidation by intact rat liver mitochondria.

The quantitative isolation of acyl-CoA esters of chain length C2-C17 from mitochondrial incubations and their analysis by reverse-phase radio-h.p.l.c. is described. Photodiode-array detection was used to characterize 2-enoyl-CoA esters. The chromatographic behaviour of all 27 intermediates of the beta-oxidation of hexadecanoyl-CoA is documented. Only C16, C14 and C12 intermediates were detected in uncoupled mitochondria oxidizing [U-14C]hexadecanoyl-CoA in the presence of fluorocitrate and carnitine, providing evidence for some organization of the enzymes of beta-oxidation [Garland, Shepherd & Yates (1965) Biochem. J. 97, 587-594; Sumegi & Srere (1984) J. Biol. Chem. 259, 8748-8752]. Rotenone increased concentrations of 3-hydroxyacyl-CoA and 2-enoyl-CoA esters and inhibited flux. These experiments provide the first direct unambiguous measurements of acyl-CoA esters in intact respiring rat liver mitochondrial fractions.     

Orientation of coenzyme A substrates, nicotinamide and active site functional groups in (Di)enoyl-coenzyme A reductases

The stereochemical course of reduction of dienoyl-coenzyme A (CoA) thiolesters catalyzed by the 2,4-dienoyl-CoA reductase from rat liver mitochondria was investigated. The configuration of the double bond in the 3-enoyl-CoA products was determined by (1)H NMR, and experiments to determine the stereochemical course of reduction at Calpha and Cdelta by use of 4-(2)H-labeled beta-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), were conducted in H(2)O and D(2)O. Defining the diastereoselectivity of the reaction, catalyzed by the Delta(3),Delta(2)-enoyl-CoA isomerase, facilitated the determination of the stereochemical course of reduction by 2, 4-dienoyl-CoA reductase. The absence of solvent exchange of the proton transferred during the Delta(3),Delta(2)-enoyl-CoA isomerase catalyzed equilibration of trans-2- and trans-3-enoyl-CoAs, coupled with the strong sequence homology to enoyl-CoA hydratase support the intramolecular suprafacial transfer of the pro-2R proton of trans-3-enoyl-CoA to the pro-4R position of trans-2-enoyl-CoA. The results indicate that the configuration of the double bond of the 3-enoyl-CoA product is trans and that a general acid-catalyzed addition of a solvent derived proton/deuteron occurs on the si face at Calpha of the dienoyl-CoA. The addition of the pro-4S hydrogen from NADPH occurs on the si face at Cdelta of trans-2, cis-4-dienoyl-CoA and on the re face at Cdelta of trans-2, trans-4-dienoyl-CoA. The stereochemical course of reduction of InhA, an enoyl-thiolester reductase from Mycobacterium tuberculosis, was also determined by use of ?4-(2)HNADH in D(2)O. The reduction of trans-2-octenoyl-CoA catalyzed by InhA resulted in the syn addition of (2)H(2) across the double bond yielding (2R,3S)-?2, 3-(2)H(2)?ctanoyl-CoA. In the crystal structure of the InhA ternary complex, the residue donating the proton to Calpha could not be identified ?Rozwarski, D. A., Vilcheze, C., Sugantino, M., Bittman, R., and Sacchettini, J. C. (1999) J. Biol. Chem. 274, 15582-15589. The current results place further restrictions on the source of the proton and suggest the reduction is stepwise.     

Purification of the multienzyme complex for fatty acid oxidation from Pseudomonas fragi and reconstitution of the fatty acid oxidation system

The multienzyme complex for fatty acid oxidation was purified from Pseudomonas fragi, which was grown on oleic acid as the sole carbon source. This complex exhibited enoyl-CoA hydratase [EC 4.2.1.17], 3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35], 3-oxoacyl-CoA thiolase [EC 2.3.1.16], cis-3,trans-2-enoyl-CoA isomerase [EC 5.3.3.3], and 3-hydroxyacyl-CoA epimerase [EC 5.1.2.3] activities. The molecular weight of the native complex was estimated to be 240,000. Two types of subunits, with molecular weights of 73,000 and 42,000, were identified. The complex was composed of two copies each of the 73,000- and 42,000-Da subunits. The beta-oxidation system was reconstituted in vitro using the multienzyme complex, acyl-CoA synthetase and acyl-CoA oxidase. This reconstituted system completely oxidized saturated fatty acids with acyl chains of from 4 to 18 carbon atoms as well as unsaturated fatty acids having cis double bonds extending from odd-numbered carbon atoms. However, unsaturated fatty acids having cis double bonds extending from even-numbered carbon atoms were not completely oxidized to acetyl-CoA: about 5 mol of acetyl-CoA was produced from 1 mol of linoleic or alpha-linolenic acid, and about 2 mol of acetyl-CoA from 1 mol of gamma-linolenic acid. These results suggested that the 3-hydroxyacyl-CoA epimerase in the complex was not operative. When the epimerase was by-passed by the addition of 2,4-dienoyl-CoA reductase to the reconstituted system, unsaturated fatty acids with cis double bonds extending from even-numbered carbon atoms were also completely degraded to acetyl-CoA.     

过氧物酶体多功能酶2中固醇载体蛋白2结构域的功能

过氧物酶体多功能酶 (包括Ⅰ型、Ⅱ型 ,简称MFE1、MFE2 )在哺乳类动物的脂类代谢中发挥其重要作用 .MFE1具有 2 烯酰CoA水合酶 1和 (3S) 羟脂酰CoA脱氢酶的活性 ,而MFE2具有 2 烯酰CoA水合酶 2和 (3R) 羟脂酰CoA脱氢酶的活性 ,两者均催化烯酰CoA在过氧物酶体β 氧化途径中的第 2步和第 3步反应 .MFE1与MFE2的氨基酸序列不具有任何同源性 ,并且它们的底物特异性也不相同 .比较哺乳类MFE1及酵母MFE2发现 ,哺乳类MFE2羧基末端带有由 12 5个残基组成的固醇载体蛋白 2 (简称SCP2 )结构域 ,其功能是未知的 .为了研究SCP2结构域在MFE2中的功能 ,将人MFE2、MFE2ΔSCP2 (删除MFE2中的SCP2 )、脱氢酶结构域、水合酶结构域以及SCP2结构域分别在E .coli中表达 ,并经纯化得到相应的重组蛋白 .通过测定 2 烯酰CoA水合酶 2和 (3R) 羟脂酰CoA脱氢酶对烯酰CoA的催化活性发现 ,带有SCP2结构域的重组蛋白的酶活力及催化效率高于删除SCP2的突变体蛋白 .实验结果表明 ,SCP2结构域可能通过增强MFE2与脂酰CoA的结合力 ,使得MFE2发挥最有效的催化活力     

Purification and properties of acyl coenzyme A thioesterase II from Rhodopseudomonas sphaeroides

A high molecular weight acyl coenzyme A (acyl-CoA) thioesterase, designated thioesterase II, has been purified 5300-fold from photoheterotrophically grown cells of Rhodopseudomonas sphaeroides. In contrast to R. sphaeroides acyl-CoA thioesterase I [Boyce, S.G., & Lueking, D.R. (1984) Biochemistry 23, 141-147], thioesterase II has a native molecular mass (Mr) of 120,000, is capable of hydrolyzing saturated and unsaturated acyl-CoA substrates with acyl chain lengths ranging from C4 to C18, and is completely insensitive to the serine esterase inhibitor diisopropyl fluorophosphate. Palmitoyl-CoA and stearoyl-CoA are the preferred (lowest Km) saturated acyl-CoA substrates and vaccenoyl-CoA is the preferred unsaturated substrate. However, comparable Vmax values were obtained with a variety of acyl-CoA substrates. Unlike a similar thioesterase present in cells of Escherichia coli [Bonner, W.M., & Bloch, K. (1972) J. Biol. Chem. 247, 3123-3133], R. sphaeroides thioesterase II displays a high ratio of decanoyl-CoA to palmitoyl-CoA activities and exhibits little ability to hydrolyze 3-hydroxyacyl-CoA substrates. Only 3-hydroxydodecanoyl-CoA supported a measurable rate of enzyme activity. With the purification of thioesterase II, the enzymes responsible for greater than 90% of the acyl-CoA thioesterase activity present in cell-free extracts of R. sphaeroides have now been identified.     

Site of inhibition of rat liver microsomal fatty acid chain elongation system by dec-2-ynoyl coenzyme A. Possible mechanism of inhibition

The present study examines the effect of the acetylenic thioester dec-2-ynoyl-CoA (delta 2 10 identical to 1-CoA) on the microsomal fatty acid chain elongation pathway in rat liver. When the individual reactions of the elongation system were measured in the presence of delta 2 10 identical to 1-CoA, the trans-2-enoyl-CoA reductase activity was markedly inhibited (Ki = 2.5 microM), whereas the activities of the condensing enzyme, the beta-ketoacyl-CoA reductase, and the beta-hydroxyacyl-CoA dehydrase were not affected. The absence of inhibition of total microsomal fatty acid elongation was attributed to the significant accumulation of the intermediates, beta-hydroxyacyl-CoA and trans-2-enoyl-CoA, without formation of the saturated elongated product, indicating that the trans-2-enoyl-CoA reductase-catalyzed reaction was the only site affected by the inhibitor. The nature of the inhibition was noncompetitive. In contrast to the delta 2 10 identical to 1-CoA, delta 3 10 identical to 1-CoA did not inhibit trans-2-enoyl-CoA reductase activity, suggesting that the mode of inhibition was not via formation of the 2,3-allene derivative. Based on the observation (a) that p-chloromercuribenzoate markedly inhibits reductase activity, (b) that dithiothreitol protects the enzyme against inactivation by delta 2 10 identical to 1-CoA, (c) of the spectral manifestation of the interaction between thiol reagents and delta 2 10 identical to 1-CoA depicting an absorbance peak similar to that of the beta-ketoacyl thioester-Mg2+ enolate complex, (d) of a similar absorbance spectrum formed by the interaction between delta 2 10 identical to 1-CoA and liver microsomes, and (e) of the absence of formation of a similar spectrum by delta 3 10 identical to 1-CoA, trans-2-10:1-CoA, or delta 2 10 identical to 1 free acid with liver microsomes, we propose that delta 2 10 identical to 1-CoA inactivates trans-2-enoyl-CoA reductase by covalently binding to a critical sulfhydryl group at or in close proximity to the active site of the enzyme.     

The effect of bovine serum albumin on partial reactions of palmitoyl-CoA chain elongation by rat liver microsomes.

In the absence of albumin, v/s curves for both condensation and overall chain elongation demonstrated that the specific activity for overall chain elongation was 3.7 times that of condensation. When the molar ratio of palmitoyl-CoA to albumin was greater than 2 : 1, the specific activity of chain elongation exceeded that of condensation. At these low albumin concentrations, in the absence of NADPH, the beta-ketostearoyl-coA was converted back to palmitate. This cleavage reaction is inhibited by albumin in a concentration-dependent manner. When the palmitoyl-CoA to albumin molar ratio was less than 2 : 1, the specific activity for condensation exceeded that for overall chain elongation and some beta-ketostearate was shown to accumulate under chain elongation conditions. The specific activity for dehydration of beta-hydroxystearoyl-CoA was maximal when the acyl-CoA to albumin molar ratio was between 10 : 1 and 4 : 1 but the rate of this reaction was not markedly influenced by variations in albumin concentration. The specific activity for the NADPH-dependent reduction of 2-trans-octa-decenoyl-CoA was 18 nmol . min(-1) . mg(-1) in the absence of albumin and increased to a maximum of 112 when the substrate to albumin molar ratio was 2 : 1. At higher albumin concentrations the reductase reaction was inhibited. Conversely, the specific activity for the reverse dehydrase was maximal at low albumin concentrations and the rate of this reaction declined as the albumin concentration increased. Our results demonstrate that albumin not only alleviates a substrate induced inhibition but also regulates the metabolic fate of 2-trans-octadecenoyl-CoA and in this regard may possibly substitute for acyl-CoA binding proteins.     

Effect of dietary alpha-linolenic acid on the activity and gene expression of hepatic fatty acid oxidation enzymes

The activities of hepatic fatty acid oxidation enzymes in rats fed linseed and perilla oils rich in alpha-linolenic acid (alpha-18:3) were compared with those in the animals fed safflower oil rich in linoleic acid (18:2) and saturated fats (coconut or palm oil). Mitochondrial and peroxisomal palmitoyl-CoA (16:0-CoA) oxidation rates in the liver homogenates were significantly higher in rats fed linseed and perilla oils than in those fed saturated fats and safflower oil. The fatty oxidation rates increased as dietary levels of alpha-18:3 increased. Dietary alpha-18:3 also increased the activity of fatty acid oxidation enzymes except for 3-hydroxyacyl-CoA dehydrogenase. Unexpectedly, dietary alpha-18:3 caused great reduction in the activity of 3-hydroxyacyl-CoA dehydrogenase measured with short- and medium-chain substrates but not with long-chain substrate. Dietary alpha-18:3 significantly increased the mRNA levels of hepatic fatty acid oxidation enzymes including carnitine palmitoyltransferase I and II, mitochondrial trifunctional protein, acyl-CoA oxidase, peroxisomal bifunctional protein, mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, 2, 4-dienoyl-CoA reductase and delta3, delta2-enoyl-CoA isomerase. Fish oil rich in very long-chain n-3 fatty acids caused similar changes in hepatic fatty acid oxidation. Regarding the substrate specificity of beta-oxidation pathway, mitochondrial and peroxisomal beta-oxidation rate of alpha-18:3-CoA, relative to 16:0- and 18:2-CoAs, was higher irrespective of the substrate/albumin ratios in the assay mixture or dietary fat sources. The substrate specificity of carnitine palmitoyltransferase I appeared to be responsible for the differential mitochondrial oxidation rates of these acyl-CoA substrates. Dietary fats rich in alpha-18:3-CoA relative to safflower oil did not affect the hepatic activity of fatty acid synthase and glucose 6-phosphate dehydrogenase. It was suggested that both substrate specificities and alterations in the activities of the enzymes in beta-oxidation pathway play a significant role in the regulation of the serum lipid concentrations in rats fed alpha-18:3.