Medium-chain Fatty acyl-CoA Research Articles

Variant of Medium-Chain Fatty Acyl-CoA Dehydrogenase Deficiency Delayed Manifestation: A Case Report

Variant of Medium-Chain Fatty Acyl-CoA Dehydrogenase Deficiency Delayed Manifestation: A Case Report

An optimized reverse β-oxidation pathway to produce selected medium-chain fatty acids in Saccharomyces cerevisiae

BackgroundMedium-chain fatty acids are molecules with applications in different industries and with growing demand. However, the current methods for their extraction are not environmentally sustainable. The reverse β-oxidation pathway is an energy-efficient pathway that produces medium-chain fatty acids in microorganisms, and its use in Saccharomyces cerevisiae, a broadly used industrial microorganism, is desired. However, the application of this pathway in this organism has so far either led to low titers or to the predominant production of short-chain fatty acids.ResultsWe genetically engineered Saccharomyces cerevisiae to produce the medium-chain fatty acids hexanoic and octanoic acid using novel variants of the reverse β-oxidation pathway. We first knocked out glycerolphosphate dehydrogenase GPD2 in an alcohol dehydrogenases knock-out strain (△adh1-5) to increase the NADH availability for the pathway, which significantly increased the production of butyric acid (78 mg/L) and hexanoic acid (2 mg/L) when the pathway was expressed from a plasmid with BktB as thiolase. Then, we tested different enzymes for the subsequent pathway reactions: the 3-hydroxyacyl-CoA dehydrogenase PaaH1 increased hexanoic acid production to 33 mg/L, and the expression of enoyl-CoA hydratases Crt2 or Ech was critical to producing octanoic acid, reaching titers of 40 mg/L in both cases. In all cases, Ter from Treponema denticola was the preferred trans-enoyl-CoA reductase. The titers of hexanoic acid and octanoic acid were further increased to almost 75 mg/L and 60 mg/L, respectively, when the pathway expression cassette was integrated into the genome and the fermentation was performed in a highly buffered YPD medium. We also co-expressed a butyryl-CoA pathway variant to increase the butyryl-CoA pool and support the chain extension. However, this mainly increased the titers of butyric acid and only slightly increased that of hexanoic acid. Finally, we also tested the deletion of two potential medium-chain acyl-CoA depleting reactions catalyzed by the thioesterase Tes1 and the medium-chain fatty acyl CoA synthase Faa2. However, their deletion did not affect the production titers.ConclusionsBy engineering the NADH metabolism and testing different reverse β-oxidation pathway variants, we extended the product spectrum and obtained the highest titers of octanoic acid and hexanoic acid reported in S. cerevisiae. Product toxicity and enzyme specificity must be addressed for the industrial application of the pathway in this organism.

Efficient production of surfactin from xylose-rich corncob hydrolysate using genetically modified Bacillus subtilis 168.

As one of the most powerful biosurfactants, surfactin has extensive application prospects in numerous industrial fields. Bacillus subtilis 168 was genetically modified to produce surfactin by increasing the supply of the precursor fatty acyl-CoA by overexpressing 4' phosphopantetheinyl transferase, medium-chain acyl-acyl carrier protein (ACP) thioesterase and fatty acyl CoA ligase (encoded by sfp, bte, and yhfL, respectively), and knocking out acyl-CoA dehydrogenase (encoded by fadE). The resulting recombinant strain BSFX022 produced 2203mg/L surfactin with xylose as carbon source. The lower accumulation of organic acids with xylose as carbon source made it possible to maintain surfactin production in a non-buffered fermentation system, and the yield reached 2074mg/L. Furthermore, to reduce the costs, waste biomass such as corncob hydrolysate and monosodium glutamate wastewater (MGW) were used, and 2032mg/L of surfactin was produced in the optimal waste-based medium. To our best knowledge, this is the first report of surfactin production using genetically modified Bacillus subtilis 168 with xylose as carbon source.

Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols.

Fatty alcohols are value-added chemicals and important components of a variety of industries, which have a >3 billion-dollar global market annually. Long chain fatty alcohols (>C12) are mainly used in surfactants, lubricants, detergents, pharmaceuticals and cosmetics while medium chain fatty alcohols (C6–C12) could be used as diesel-like biofuels. Microbial production of fatty alcohols from renewable feedstock stands as a promising strategy to enable sustainable supply of fatty alcohols. In this study, we report, for the first time, that medium chain fatty alcohols could be produced in yeast via targeted expression of a fatty acyl-CoA reductase (TaFAR) in the peroxisome of Saccharomyces cerevisiae. By tagging TaFAR enzyme with peroxisomal targeting signal peptides, the TaFAR could be compartmentalized into the matrix of the peroxisome to hijack the medium chain fatty acyl-CoA generated from the beta-oxidation pathway and convert them to versatile medium chain fatty alcohols (C10 & C12). The overexpression of genes encoding PEX7 and acetyl-CoA carboxylase further improved fatty alcohol production by 1.4-fold. After medium optimization in fed-batch fermentation using glucose as the sole carbon source, fatty alcohols were produced at 1.3 g/L, including 6.9% 1-decanol, 27.5% 1-dodecanol, 2.9% 1-tetradecanol and 62.7% 1-hexadecanol. This work revealed that peroxisome could be engineered as a compartmentalized organelle for producing fatty acid-derived chemicals in S. cerevisiae.

DarR, a TetR-like Transcriptional Factor, Is a Cyclic Di-AMP-responsive Repressor in Mycobacterium smegmatis

Cyclic dinucleotides, including cyclic di-AMP (c-di-AMP), are known to be ubiquitous second messengers involved in bacterial signal transduction. However, no transcriptional regulator has been characterized as a c-di-AMP receptor/effector to date. In the present study, using a c-di-AMP/transcription factor binding screen, we identified Ms5346, a TetR family regulator in Mycobacterium smegmatis, as a c-di-AMP receptor in bacteria. Ms5346 could specifically bind c-di-AMP with K(d) of 2.3 ± 0.5 μM. Using EMSA and DNase I footprinting assays, c-di-AMP was found to stimulate the DNA binding activity of Ms5346 and to enhance its ability to protect its target DNA sequence. A conserved 14-bp palindromic motif was identified as the DNA-binding site for Ms5346. Further, Ms5346 was found to negatively regulate expression of three target genes including Ms5347 (encoding a major facilitator family transporter), Ms5348 (encoding a medium chain fatty acyl-CoA ligase), and Ms5696 (encoding a cold shock protein, CspA). Ms5346 is the first cyclic di-AMP receptor regulator to be identified in bacteria, and we have designated it as DarR. Our findings enhance our understanding of the function and regulatory mechanism of the second messenger c-di-AMP in bacteria.

Overexpression of the apple alcohol acyltransferase gene alters the profile of volatile blends in transgenic tobacco leaves

Alcohol acyltransferases (AATs) are key enzymes in ester biosynthesis. Previous studies have found that AAT may be a stress-related gene. To investigate further the function of the apple alcohol acyltransferase gene (MdAAT2), transgenic tobacco plants overexpressing MdAAT2 were generated. Gas chromatography-mass spectroscopy analysis showed that the volatile blends were altered in these transgenic tobacco leaves. Although no apple-fruity volatile esters were detected in transgenic tobacco leaves, methyl caprylate, methyl caprate, and methyl dodecanoate were newly generated, and the concentrations of methyl benzoate and methyl tetradecanoate were significantly increased, suggesting that MdAAT2 may use medium-chain fatty acyl CoA and benzoyl-CoA as acyl donors together with methanol acceptors as substrates. Surprisingly, the concentrations of linalool were significantly increased in transgenic tobacco leaves, which may mediate the repellent effect on Myzus persicae (Sulzer) aphids. Using methyl jasmonate (MeJA) and wounding treatments, we found that MdAAT2 may substitute for the partial ability of MeJA to induce the production of linalool in transgenic plants. These data suggest that MdAAT2 may be involved in the response to the MeJA signal and may play a role in the response to biotic and abiotic stress.

Molecular Identification of a Novel Mammalian Brain Isoform of Acyl-CoA:Lysophospholipid Acyltransferase with Prominent Ethanolamine Lysophospholipid Acylating Activity, LPEAT2

Acyl-CoA-dependent lysophospholipid acyltransferases play an important role in attaining the appropriate molecular species of phospholipids. A number of genes encoding these activities were recently identified. It has become clear that multiple genes can encode one enzymatic activity and that a given gene may encode multiple activities. Here we report the identification of a gene encoding a mammalian acyl-CoA-dependent lysophospholipid acyltransferase with prominent activity toward ethanolamine-containing lysophospholipids, which we termed acyl-CoA:lysophosphatidylethanolamine acyltransferase 2, LPEAT2 (previously annotated as AYTL3 or AGPAT7). LPEAT2 is predominantly expressed in brain, coinciding with an enrichment of phosphatidylethanolamine in this tissue. Ectopic expression of LPEAT2 in mammalian HEK293T cells led to a dramatic increase (up to 9-fold) in LPEAT activity when compared with cells transfected with empty vector or an unrelated acyltransferase. LPEAT2 also exhibited significant acyl-CoA-dependent acyltransferase activity toward 1-O-alkenyl-lysophosphatidylethanolamine, lysophosphatidylglycerol, 1-O-alkyl-lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylcholine but lacked appreciable acylating activity toward glycerol 3-phosphate, lysophosphatidic acid, lysophosphatidylinositol, and diacylglycerol, demonstrating multiple but selective functions of LPEAT2 as an enzyme involved in phospholipid remodeling. LPEAT2 recognizes a broad range of medium and long chain fatty acyl-CoA, and its activity was not affected by Ca(2+). When overexpressed in mammalian cells, LPEAT2 is localized to the endoplasmic reticulum. siRNA-mediated knockdown of LPEAT2 in HEK293T cells significantly decreased LPEAT and 1-alkenyl-LPEAT activities but did not affect other lysophospholipid acylating activities. These findings identify LPEAT2 as an important enzyme in the biosynthesis of ethanolamine-containing phospholipids, especially in brain.

Hypothalamic malonyl-CoA triggers mitochondrial biogenesis and oxidative gene expression in skeletal muscle: Role of PGC-1alpha.

Previous investigations show that intracerebroventricular administration of a potent inhibitor of fatty acid synthase, C75, increases the level of its substrate, malonyl-CoA, in the hypothalamus. The "malonyl-CoA signal" is rapidly transmitted to skeletal muscle by the sympathetic nervous system, increasing fatty acid oxidation, uncoupling protein-3 (UCP3) expression, and thus, energy expenditure. Here, we show that intracerebroventricular or intraperitoneal administration of C75 increases the number of mitochondria in white and red (soleus) skeletal muscle. Consistent with signal transmission from the hypothalamus by the sympathetic nervous system, centrally administered C75 rapidly (< or =2 h) up-regulated the expression (in skeletal muscle) of the beta-adrenergic signaling molecules, i.e., norepinephrine, beta3-adrenergic receptor, and cAMP; the transcriptional regulators peroxisomal proliferator activator regulator gamma coactivator 1alpha (PGC-1alpha) and estrogen receptor-related receptor alpha (ERRalpha); and the expression of key oxidative mitochondrial enzymes, including pyruvate dehydrogenase kinase, medium-chain length fatty acyl-CoA dehydrogenase, ubiquinone-cytochrome c reductase, cytochrome oxidase, as well as ATP synthase and UCP3. The role of PGC-1alpha in mediating these responses in muscle was assessed with C2C12 myocytes in cell culture. Consistent with the in vivo response, adenovirus-directed expression of PGC-1alpha in C2C12 muscle cells provoked the phosphorylation/inactivation and reduced expression of acetyl-CoA carboxylase 2, causing a reduction of the malonyl-CoA concentration. These effects, coupled with an increased carnitine palmitoyltransferase 1b, led to increased fatty acid oxidation. PGC-1alpha also increased the expression of ERRalpha, PPARalpha, and enzymes that support mitochondrial fatty acid oxidation, ATP synthesis, and thermogenesis, apparently mediated by an increased expression of UCP3.

Facile and restricted pathways for the dissociation of octenoyl-CoA from the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-FADH2-octenoyl-CoA charge-transfer complex: energetics and mechanism of suppression of the enzyme's oxidase activity.

In a previous paper, we demonstrated that the reductive half-reaction of medium-chain fatty acyl-CoA dehydrogenase (MCAD), utilizing octanoyl-CoA as physiological substrate, generates two (kinetically distinct) forms of the reduced enzyme (MCAD-FADH2) - octenoyl-CoA charge-transfer complexes [Kumar, N.R., & Srivastava, D.K. (1994) Biochemistry 33, 8833-8841]. We present evidence that octenoyl-CoA dissociates from the second (most stable) charge-transfer complex (referred to as CT2) via two alternative ("facile" and "restricted") pathways. The dissociation of octenoyl-CoA via the facile pathway involves the reversal of the overall reductive half-reaction of the enzyme, generating MCAD-FAD - octanoyl-CoA as the Michaelis complex, followed by dissociation of the latter complex into MCAD-FAD + octanoyl-CoA. Hence, via this pathway, octenoyl-CoA is released from the enzyme site in the form of octanoyl-CoA. In contrast, the restricted pathway involves a direct (albeit slow) dissociation of octenoyl-CoA from CT2 to yield MCAD-FADH2 + octenoyl-CoA. The kinetic profile for the dissociation of octenoyl-CoA via the restricted pathway matches the rate of oxidation of the reduced flavin (within CT2) by O2. This suggests that the oxidase activity of the enzyme remains suppressed as long as the reduced enzyme predominates in the form of the charge-transfer complex(es). The oxidase activity of the enzyme emerges concomitantly with the conversion of CT2 to the MCAD-FADH2 - octenoyl-CoA Michaelis complex. The energetic basis for the dissociation of octenoyl-CoA via the facile and restricted pathways and the mechanism of suppression of the oxidase activity of the enzyme are discussed.

Effect of transition-state analogues on the redox properties of medium-chain acyl-CoA dehydrogenase.

The binding of substrate/product or transition-state intermediates modifies the properties of medium-chain fatty acyl-CoA dehydrogenase (MCAD) by causing the redox potential to shift positive and the oxygen reactivity to slow by 3000-fold. Two ligands, identified as being the most effective in slowing oxygen reactivity, were 2-azaoctanoyl-CoA and 3-thiaoctanoyl-CoA [Wang, R., & Thorpe, C. (1991) Biochemistry 30, 7895-7901]. We have measured the potential shifts caused by the binding of both ligands to determine which is most similar to the potential shift caused by substrate/product mixture, the assumption being that the best transition-state structural intermediate would give the potential shift most similar to that of substrate/product [Lenn, N.D., Stankovich, M.T., & Liu, H. (1990) Biochemistry 29, 10594-10602]. Both ligands shifted the potential positive, but the shift caused by 2-azaoctanoyl-CoA was 65% that of substrate/product, while 3-thiaoctanoyl-CoA was only 20% of that value. This positive shift is proposed to be caused by a resonance form stabilized by the interaction of the catalytically essential carbonyl of the acyl-CoA with two hydrogen bonds from the enzyme, which induces a partial negative charge on the carbonyl and a partial positive charge on carbon 2 of the ligand and carbon 3 of the substrate/product couple. The X-ray structure shows that carbons 2 and 3 of the substrate/product overlap the diazadiene portion of the flavin ring [Kim, J.-J. P., Wang, M., & Paschke, R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7523-7527].(ABSTRACT TRUNCATED AT 250 WORDS)

"Dehydrogenase" and "Oxidase" Reactions of Medium-Chain Fatty Acyl-CoA Dehydrogenase Utilizing Chromogenic Substrates: Role of the 3',5'-Adenosine Diphosphate Moiety of the Coenzyme A Thioester in Catalysis

We undertook a comparative investigation of the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction utilizing indole-, furyl-, and 4-(dimethylamino)phenyl-substituted propionyl- and acryloyl-CoAs as potential substrate/product pairs. All these propionyl-CoA derivatives undergo MCAD-catalyzed conversion into their corresponding acryloyl-CoAs via both "dehydrogenase" (in the presence of "organic" electron acceptors) and "oxidase" (buffer-dissolved oxygen serving as the electron acceptor) pathways [Johnson, J. K., Wang, Z. X., & Srivastava, D. K. (1992) Biochemistry 31, 10564-10575]. The steady-state kinetic parameters for the enzyme utilizing these substrates reveal that the KmS (for the CoA substrates) and kcatS for the dehydrogenase reaction are at least an order of magnitude higher than those for the oxidase reaction. As with the CoA substrates, the enzyme catalyzes the conversion of indolepropionyl pantetheine phosphate (IPPP) into indoleacryloyl pantetheine phosphate (IAPP) via these two pathways. However, with IPPP as substrate, the Km (for IPPP) and kcat values of the dehydrogenase and oxidase reactions are the same. These, coupled with the spectral changes of the enzyme-product complexes as well as the binding affinities of the enzyme-substrate/product complexes, lead to the following conclusions: (1) The aromatic/heterocyclic group-containing substrates are converted into their corresponding products via both the dehydrogenase and the oxidase pathways. (2) The 3',5'-ADP moiety of the CoA thioester provides a significant fraction of the total binding energy in stabilizing the enzyme-substrate/product complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Reductive half-reaction of medium-chain fatty acyl-CoA dehydrogenase utilizing octanoyl-CoA/octenoyl-CoA as a physiological substrate/product pair: similarity in the microscopic pathways of octanoyl-CoA oxidation and octenoyl-CoA binding.

Of the different chain length fatty acyl-CoA substrates, octanoyl-CoA has been known as one of the most efficient (and physiological) substrates for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction of MCAD-FAD with octanoyl-CoA ([MCAD-FAD] << [octanoyl-CoA]), measured via the stopped-flow technique, at 5 degrees C was characterized by a biphasic decrease and increase in absorptions at 450 and 545 nm, respectively. The average values of the fast (1/tau 1) and slow (1/tau 2) relaxation rate constants, derived from the data at these wavelengths, were found to be 319.7 +/- 33.5 and 28.8 +/- 12.5 s-1, respectively, and both of these relaxation rate constants remained invariant between 8 and 200 microM concentrations of octanoyl-CoA. Under identical experimental conditions, we measured time courses for the interaction of MCAD-FAD with octenoyl-CoA ([MCAD-FAD] << [octenoyl-CoA]) by monitoring the absorption changes at 299, 394, and 440 nm. The binding profile was consistent with a biphasic decrease (at 440 nm) and increase (at 299 and 394 nm) in absorbance, with similar magnitudes of fast [1/tau 1 (average) = 382.3 +/- 39.8 s-1] and slow [1/tau 2 (average) = 14.3 +/- 7.4 s-1] relaxation rate constants. The observed relaxation rate constants were, once again, found to be invariant with changes in the octenoyl-CoA concentration from 40 to 150 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular basis of the medium-chain fatty acyl-CoA dehydrogenase-catalyzed "oxidase" reaction: pH-dependent distribution of intermediary enzyme species during catalysis.

In a previous paper, we demonstrated that the medium-chain fatty acyl-CoA dehydrogenase-catalyzed (MCAD-catalyzed) reductive half-reaction of indolepropionyl-CoA proceeds via formation of a chromophoric intermediary species "X" (absorption maximum = 400 nm) and proposed that the decay of this species might limit the overall rate of the "oxidase" reaction [Johnson, J. K., & Srivastava, D. K. (1993) Biochemistry 32, 8004-8013]. During this latter reaction, the buffer-dissolved O2 served as an electron acceptor [Johnson, J. K., Wang, Z. X., & Srivastava, D. K. (1992) Biochemistry 31, 10564-10575]. To ascertain whether the intrinsic stability of X influences the oxidase activity, we undertook a detailed kinetic investigation of this enzyme at different pH values. The time-resolved spectra for the reductive half-reaction (obtained via the rapid-scanning stopped-flow method) at different pH values reveal that the amplitude of the intermediary (X) spectral band is more pronounced at a lower pH (pH 6.4) than at a higher pH (pH 9.0). Single-wavelength transient kinetic data for the reductive half-reaction (in both the forward and the reverse direction) at all pH values are consistent with fast (1/tau 1) and slow (1/tau 2) relaxation rate constants. Of these, whereas the fast relaxation rate constant for the reaction in the forward direction (1/tau 1f) decreases with an increase in pH, the corresponding slow relaxation rate constant (1/tau 2f) increases with an increase in pH. The pH-dependent steady-state kinetic data reveal that, like 1/tau 2f, kcat for the MCAD-catalyzed oxidase reaction increases with an increase in the pH of the buffer media.(ABSTRACT TRUNCATED AT 250 WORDS)

Corrections - Mechanistic Investigation of Medium-Chain Fatty Acyl-CoA Dehydrogenase Utilizing 3-Indolepropionyl/Acryloyl-CoA as Chromophoric Substrate Analogues

Corrections - Mechanistic Investigation of Medium-Chain Fatty Acyl-CoA Dehydrogenase Utilizing 3-Indolepropionyl/Acryloyl-CoA as Chromophoric Substrate Analogues

Detection and identification of a chromophoric intermediate during the medium-chain fatty acyl-CoA dehydrogenase-catalyzed reaction via rapid-scanning UV/visible spectroscopy.

We have investigated the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction via rapid-scanning stopped-flow (RSSF) UV/vis spectroscopy, combined with the single-wavelength stopped-flow technique, utilizing 3-indolepropionyl-CoA (IPCoA) and trans-3-indoleacryloyl-CoA (IACoA) as chromophoric pseudosubstrates. The RSSF spectral data reveal that formation of an intermediary species with an absorbance maximum at 400 nm and a broad charge-transfer band around 600 nm accompanies the reduction of MCAD-FAD by IPCoA. In the presence of high concentrations of enzyme ([MCAD] >> [IPCoA]) the intermediary spectral band at 400 nm remains unperturbed, whereas in the presence of low concentrations of enzyme ([MCAD] << [IPCoA]) it slowly shifts to an absorption band with an absorbance maximum at 370 nm. Appearance and disappearance of this intermediary species coincides with the appearance and disappearance of the charge-transfer band. Single-wavelength stopped-flow studies, performed under similar high and low enzyme conditions, were consistent with one (1/tau 1) and two (1/tau 1 > 1/tau 2) relaxation rate constants, respectively. These findings, combined with relaxation studies performed in the reverse directions as well as substrate and product binding studies with the oxidized and reduced forms of the enzyme, have allowed us to conclude the following: (1) the intermediary species possesses the properties of reduced flavin and highly conjugated reaction product IACoA (absorbance maximum = 400 nm); (2) this intermediary species collapses into an MCAD-FADH2-IACoA complex (absorbance maximum = 370 nm) in the presence of excessive concentrations of IPCoA; the collapse is being driven by the competitive binding of IPCoA with the reduced form of the enzyme; (3) the 400-nm absorption band and the charge-transfer band are given by the same intermediary species formed during the enzyme-catalyzed reaction pathway. The role of protein conformational changes in modulating the substrate/product structures during the MCAD-catalyzed reaction is discussed.

Renal Handling of Carnitine in Secondary Carnitine Deficiency Disorders

Reduced plasma and tissue concentrations of carnitine, a cofactor required for fatty acid oxidation, are present in patients with inherited disorders of mitochondrial acyl-CoA oxidation that are associated with accumulations of acylcarnitines. To determine whether the secondary carnitine deficiency in these patients is due to excessive urinary loss of acylcarnitines, the development of carnitine deficiency was examined in patients with four different acyl-CoA oxidation disorders, including medium-chain and long-chain fatty acyl-CoA dehydrogenase deficiencies, isovaleric acidemia, and propionic acidemia. After a 3-mo period of treatment with oral carnitine to raise plasma total carnitine concentrations to or above normal, patients were started on a carnitine-free diet and the changes in plasma total and free carnitine levels and urinary total and free carnitine excretion were followed for 5 d. Patients with all four disorders showed a return of plasma carnitine levels and urinary carnitine excretion to baseline within 2 to 4 d. The rapidity of these changes could not be explained solely by excessive acylcarnitine wasting. Continued excretion of free carnitine in all patients indicated the additional presence of an impairment in renal transport of free carnitine. Consistent with this interpretation, estimates of renal thresholds for free carnitine gave values that were less than that for a control child in all four disorders and ranged as low as one half those reported in normal individuals. These results suggest that secondary carnitine deficiency in the acyl-CoA oxidation disorders is due to indirect as well as direct effects of accumulated acylcarnitines.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and sequencing of thioesterase B cDNA and stimulation of expression of the thioesterase B gene associated with hormonal induction of peroxisome proliferation

The primary structure deduced from the cDNA of a medium-chain fatty acyl-CoA hydrolase designated thioesterase B from the uropygial gland of mallard duck was determined. A near full-length thioesterase B cDNA was isolated from a duck uropygial gland cDNA library using a 120-base pair polymerase chain reaction probe generated from first strand of cDNA and primers synthesized on the basis of two segments of the enzyme. The nucleotide sequence of this cDNA showed an open reading frame encoding a polypeptide of 557 amino acids including a 25-amino acid leader sequence. It showed little homology to the thioesterase domain of fatty acid synthase and S-acyl fatty acid synthase thioesterase, but showed homology to some esterases such as carboxylesterases. Northern blot showed one major transcript at 2.4 kilobases. The highest level of this transcript was in the uropygial gland, much less in the liver and kidney, and not detectable in other organs. Elevation of thioesterase B transcript level was associated with peroxisome proliferation occurring in the mating season and the increase in transcript level correlated with peroxisome proliferation and synthesis of 3-hydroxyfatty acid diester pheromones resulting from estradiol treatment. This thioesterase may be associated with peroxisome proliferation or peroxisomal metabolism.

Microscopic pathway for the medium-chain fatty acyl CoA dehydrogenase catalyzed oxidative half-reaction: changes in the electronic structures of flavin and CoA derivatives during catalysis.

In a previous communication, we demonstrated that the medium-chain fatty acyl CoA dehydrogenase (MCAD) catalyzed conversion of 3-indolepropionyl CoA (IPCoA) to trans-3-indoleacryloyl CoA (IACoA) proceeds via the formation of an intermediary species X that possesses the electronic properties of reduced flavin and highly conjugated CoA product. Since the steady-state turnover of the enzyme-catalyzed dehydrogenation reaction precisely matches with the rate of formation of X [Johnson, J. K., & Srivastava, D. K. (1993) Biochemistry 32, 8004-8013], the latter species appeared to be the likely site for the transfer of electrons to external electron acceptors (e.g., ferricenium hexafluorophosphate, FcPF6). To probe the microscopic pathway for the oxidative half-reaction, we employed a sequential mixing stopped-flow technique utilizing IPCoA as the enzyme substrate and FcPF6 as the electron acceptor. The time-dependent changes in absorption at 450, 415, and 367 nm were measured upon mixing FcPF6 with previously mixed and aged solutions of MCAD-FAD+IPCoA in the stopped-flow syringes. The kinetic traces show an increase (1/tau 1) followed by a decrease (1/tau 2) in absorption at 450 and 415 nm, and a lag (corresponding to the time regime of 1 u 1) followed by an increase in absorption (1/tau 2) at 367 nm. The relaxation rate constants (1/tau's) thus measured remain unaffected, with variations in the aging time; however, the amplitudes of these phases increase up to the aging time of 5 s, after which the amplitudes attain maxima. For an aging time of 5 s, 1/tau 1 and 1/tau 2 show a linear and a hyperbolic dependence on the FcPF6 concentration, respectively. These, coupled with the complementary studies involving butyryl CoA as a nonchromophoric substrate for this enzyme, lead us to propose the following sequence of events during the MCAD-catalyzed oxidative half-reaction: (1) The enzyme-catalyzed oxidative half-reaction proceeds via the formation of a collision complex between X and FcPF6 during the fast (1/tau 1) relaxation phase. (2) The reduced flavin moiety of X is oxidized via (rapid) transfer of electrons to FcPF6 within the collision complex, without formation of a detectable (metastable) flavin semiquinone intermediate. (3) The transfer of electrons is accompanied by changes in the electronic structures of both the flavin and IACoA moieties within the enzyme-IACoA complex. The electronic structure of this newly formed complex is exactly the same as that formed upon isomerization of the MCAD-FAD-IACoA complex [Johnson, J. K., Wang, Z. X., & Srivastava, D. K. (1992) Biochemistry 31, 10564-10575].(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing (3-indolpropionyl/acryloyl-CoA as chromophoric substrate analogs

The CoA derivative 3-indolepropionyl-CoA (IPCoA) serves as a competent pseudosubstrate for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction product trans-3-indoleacryloyl-CoA (IACoA) exhibits a characteristic UV-vis absorption spectrum with lambda max = 367 nm and epsilon 367 = 26,500 M-1 cm-1. The chromophoric nature of IACoA allows us to measure the direct conversion of substrate to product (at 367 nm) without recourse to absorption signals for either the enzyme-bound flavin or the coupling electron acceptors, as well as probe the enzyme site environment. The interaction of IACoA with medium chain fatty acyl-CoA dehydrogenase (MCAD)-FAD is characterized by resultant (spectra of the mixture minus the individual components) absorption peaks at 490, 417, and 355 nm. These absorption peaks increase in magnitude as the pH of the buffer media decreases. Transient kinetic analysis for the interaction of MCAD-FAD with IACoA suggests that the formation of the enzyme-IACoA complex proceeds in two steps. The first (fast) step involves the formation of an E-IACoA collision complex, which [formula: see text] is isomerized (concomitant with changes in the protein structure) to an E*-IACoA complex in the second (slow) step. We have studied the effect of pH on Kc, k2, and k-2. While Kc shows practically no dependence on pH (within a 2-fold variation between pH 6.0 and 9.5), k2 and k-2 show a strong dependence on pH. Both k2 and k-2 exhibit a sigmoidal dependence on the pH of the buffer media, with pKa's of 7.53 and 8.30, respectively. In accordance with the model presented herein, the pKa of 7.53 represents an enzyme site group which is involved in the interaction with IACoA within the E-IACoA collision complex. This pKa is perturbed to 8.30 upon isomerization of the collision complex. The pH-dependent changes in k2 and k-2 are such that the equilibrium distribution between E-IACoA and E*-IACoA is favored to the latter complex (by about 20-fold) at lower pH than at higher pH. A cumulative account of the spectral, kinetic, and thermodynamic properties of the enzyme-IACoA complexes has allowed us delineate the microscopic pathway by which the E-IACoA isomerization (presumably via protein conformational changes) is coupled to the proton equilibration steps.

Enzymes of fatty acid beta-oxidation in developing brain.

Developmental profiles were determined for the activities of eight enzymes involved in fatty acid beta-oxidation in rat brain. The enzymes studied were the palmitoyl-CoA, octanoyl-CoA, butyryl-CoA, glutaryl-CoA, and 3-hydroxyacyl-CoA dehydrogenases, the enoyl-CoA hydratase (crotonase), and the C4- and C10-thiolases. With the exception of the thiolases, all of the activities (expressed on the basis of brain weight) increased during the postnatal period of brain maturation. The activity of octanoyl-CoA dehydrogenase was elevated markedly compared to that of palmitoyl-CoA dehydrogenase at all developmental stages and in all brain regions in the rat. A similar relationship between these enzymes was observed in various regions of adult human brain. Comparisons of the activities of the beta-oxidation enzymes in human brain versus human skeletal muscle and in cultured neural cell lines (neuroblastoma and glioma) versus cultured skin fibroblasts revealed that the elevated activity of octanoyl-CoA dehydrogenase relative to palmitoyl-CoA dehydrogenase was specific to the neural tissues. This relationship was particularly evident when the enzyme activities were normalized to the activity of crotonase. The data support previous findings with radiochemical tracers, indicating that the brain is capable of utilizing fatty acids as substrates for oxidative energy metabolism. The relatively high activity of the medium-chain fatty acyl-CoA dehydrogenase in neural tissue may represent an adaptive mechanism to protect the brain from the known encephalopathic effects of octanoate and other medium-chain fatty acids that readily cross the blood-brain barrier.