9,638 research outputs found
The dynamic organization of fungal acetyl-CoA carboxylase
No full textAcetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control
Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase
No full text(R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl-CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by β-ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl-CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl-CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g L^[-1] (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g L^[-1], with a productivity of 0.22 g L^[-1] h^[-1]. The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of 13C carbonyl carbon labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB
Molecular regulation of the cardiac-enriched acetyl-CoA carboxylase isoform (ACCβ) : a novel target for therapeutic interventions in cardiovascular disease
No full textIncludes bibliographical references (leaves 130-173).Metabolic remodeling is thought to be an important contributor towards the development of various cardiac pathophysiologic conditions. Therefore, studies attempting to delineate undenying mechanisms driving cardiac metabolic remodeling represent an important initiative toward the development of novel therapeutic interventions. To further investigate the role of metabolic substrate switches in the heart, we focused on a pivotal, rate-limiting step of cardiac fatty acid metabolism i.e. an upstream modulator of long-chain fatty acid importation into the mitochondrion. In the heart, long-chain fatty acids are transported into the mitochondrion by the rate-limiting enzyme, carnitine palmitoyl transferase 1 (CPT1). CPT1 is potently inhibited by malonyl-CoA, the product of the acetylCoA carboxylation reaction that is catalyzed by acetyl-CoA carboxylase (ACC). Recent studies have demonstrated that metabolic fuels such as fatty acids and glucose can function as signaling ligands, directing transcriptional regulation of numerous metabolic genes. However, transcriptional mechanisms directing the gene expression of the cardiac isoform of acetyl-CoA carboxylase (ACCβ) are less well understood. Previously, four E-box (CANNTG) sequence motifs were identified on the human ACCβ promoter. Since E-boxes act as binding sites for upstream stimulatory factors (US F), putative glucose-responsive transcriptional modulators, we hypothesized that ACCβ is induced by USF1 in a glucosedependent manner. To investigate this, we began by acutely fasting and subsequently refeeding Balb/C mice with a carbohydrate-enriched diet. Here, high carbohydrate feeding resulted in elevated systemic glucose levels associated with increased cardiac ACCβ gene and protein expression. To further explore these interesting findings, we tranSiently cotransfected neonatal card iom yocytes , H9C2 myoblasts, CV-1 fibroblasts and HepG2 hepatocytes with the full-length and deletion constructs of the human ACC[3 gene promoter together with a putative activator and repressor expression vector, respectively: a) USF1 (glucose-responsive transcription factor) - the rationale that it should elevate ACCβ gene promoter activity in accordance with the glucose-fatty acid cycle, and b) nuclear respiratory factor 1(NRF1) - the hypothesis being that this mitochondrial biogenesis and β-oxidation enhancing modulator would be expected to attenuate ACCβ promoter activity in order to increase fatty acid oxidation capacity
Six amino acid substitutions in the carboxyl-transferase domain of the plastidic acetyl-CoA carboxylase gene are linked with resistance to herbicides in a Lolium rigidum population
No full text• The molecular basis of an acetyl-CoA carboxylase (ACCase) target-based resistant Lolium rigidum population (WLR 96) was studied here. • The carboxyl-transferase domain of the plastidic ACCase gene from resistant individuals was amplified by PCR and sequenced. The DNA sequences were aligned and compared with a susceptible population. • Six amino acid substitutions were identified in the resistant population. The substitution Ile-2041-Asn, known to confer resistance to ACCase-inhibiting herbicides aryloxyphenoxypropionate (APP) in Alopecurus myosuroides, was identified in most resistant plants but it is always linked with other amino acid substitutions. This was confirmed by a cleaved amplified polymorphism (CAP) marker and an allele-specific PCR. The sole amino acid substitution Ile-2041-Asn was not found in this population. It is likely this mutation evolved later among individuals already possessing the other substitutions. Three haplotypes were identified from the resistant population based on the six amino acid combinations, and two are linked with herbicide resistance in this population. • The multiple amino acid substitutions including the Ile-2041-Asn form the molecular basis endowing a high degree of resistance to ACCase-inhibiting herbicides in this L. rigidum population
Determination Of R-3-Hydroxyacyl-ACP-COA Transferase (PhaG) Structure
No full textR-3-hydroxyacyl-ACP-CoA transferase (PhaG) catalyzes the conversion of (R)-3-
hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA derivatives, which serve as the
ultimate precursor for polyhydroxyalkanoate (PHA) polymerization from unrelated
substrates. R-3-hidroksiasil-ACP-CoA tranferase (PhaG) berfungsi sebagai pemangkin
penukaran (R)-3-hidroksiasil-ACP kepada (R)-3-hidroksiasil-CoA, sebagai
prekursor utama untuk pempolimeran polyhydroxyalkanoate (PHA) daripada
substrat tidak berkaitan
Molecular basis and functional characterization of human 3-methylcrotonyl-CoA carboxylase deficiency
No full text3-Methylcrotonyl-CoA carboxylase (MCC) deficiency is a rare disorder of leucine catabolism inherited as an autosomal recessive trait. The phenotypic expression of the disease is highly variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. Most patients, however, are asymptomatic until an episode of acute metabolic decompensation following intercurrent illness leads to the diagnosis. The metabolic phenotype of MCC deficiency includes a characteristic organic aciduria with greatly increased excretion of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine and elevated blood levels of 3-hydroxyisovalerylcarnitine, usually in combination with severe secondary carnitine deficiency due to urinary loss of carnitine esters. Introduction of tandem mass spectrometry (TMS) to newborn screening has revealed an unexpectedly high incidence of MCC deficiency and indicates that this disorder is the most frequent organic aciduria.
MCC is one of the four biotin-dependent carboxylases present in humans. MCC is a mitochondrial enzyme composed of biotin containing subunits and smaller subunits, encoded by MCCA and MCCB, respectively. In order to understand the molecular biology of human MCC, including the molecular defects causing MCC deficiency and their functional consequences, MCC-deficient patients were studied and attempts were made to correlate molecular defects with the phenotypic variability in our patient population.
In this thesis mutation analysis is described in 30 MCC-deficient probands, of whom 10 came to attention because of clinical symptoms, 18 were asymptomatic newborns detected by TMS based newborn screening, and 2 were affected but asymptomatic mothers diagnosed by abnormal metabolites in the neonatal screening samples of their healthy babies. Among these 30 probands, 11 have mutations in MCCA, and 19 in MCCB. We identified 10 novel MCCA and 14 novel MCCB mutant alleles including missense, nonsense, frameshift and splice site mutations.
In order to confirm the functional consequences of MCCA and MCCB missense mutations, we used transient transfection of SV40T-transformed MCCA and MCCB deficient skin fibroblasts to express 1 MCCA and 3 MCCB missense alleles. The MCCB missense alleles expressed showed no or very low residual activity, whereas the MCCA missense allele had 26% residual activity of wild type, thus confirming that 3 out of 4 missense alleles expressed have a deleterious effect on enzyme activity. The apparent severity of MCC mutations contrasts with the variety of clinical phenotypes found in MCC-deficient patients. Our data demonstrate no clear correlation between genotype and phenotype suggesting that factors other than the genotype at the MCC loci have a major influence on the phenotype of MCC deficiency.
To analyse the MCC polypeptides under steady-state condition in fibroblasts of MCCA deficient patients, we carried out Western blot analysis. Our results demonstrate that the MCC protein was absent in 5 patients homozygous or compound heterozygous for nonsense or frameshift mutations that are expected to result in a truncated protein. The MCC protein was also absent in 2 further compound heterozygous patients in whom only one missense allele could be identified in the RT-PCR products. The second allele could not be detected because presumably this mutant allele is unstable and degraded by the mechanisms of nonsense-mediated mRNA decay. In contrast, Western blot analysis of the MCC protein was normal in amount and size in 4 patients carrying MCCA-p.R385S. This is in agreement with previous studies, which show that the protein product of this allele is stable.
Finally, we provide evidence that the missense mutation MCCA-p.R385S has a dominant negative effect on the activity of wild type. Biotin can partially reverse this negative effect and result in biotin responsiveness in vivo. This is the first example of biotin responsiveness for an isolated partial deficiency of any of the biotin-dependent carboxylases, suggesting that therapeutic trials with biotin in patients carrying this mutant allele are warranted
CoA and fatty acyl-CoA derivatives mobilize calcium from a liver reticular pool
No full textThe effect of CoA and fatty acyl-CoA esters on Ca2+ fluxes has been studied in isolated liver microsomes and in digitonin-permeabilized hepatocytes. When microsomes were loaded with increasing concentrations of Ca2+ (6-29 nmol/mg of protein), the extent to which CoA and palmitoyl-CoA released Ca2+ increased. At 23 nmol of Ca2+/mg of protein, half-maximal [CoA] and [palmitoyl-CoA] were 35 and 50 μM respectively. Under conditions of minimal Ca2+ loading, net release of Ca2+ was absent, but Ca2+ translocation from a CoA-sensitive to a CoA-insensitive pool took place. The effect of CoA required the presence of fatty acids, probably to form fatty acyl esters. In permeabilized hepatocytes, the pool(s) mobilized by CoA (or by palmitoyl-CoA) appeared to be different from that mobilized by Ins(1,4,S)P3
Succinyl-CoA:( <i>R</i> )-Benzylsuccinate CoA-Transferase: an Enzyme of the Anaerobic Toluene Catabolic Pathway in Denitrifying Bacteria
No full textABSTRACT
Anaerobic microbial toluene catabolism is initiated by addition of fumarate to the methyl group of toluene, yielding (
R
)-benzylsuccinate as first intermediate, which is further metabolized via β-oxidation to benzoyl-coenzyme A (CoA) and succinyl-CoA. A specific succinyl-CoA:(
R
)-benzylsuccinate CoA-transferase activating (
R
)-benzylsuccinate to the CoA-thioester was purified and characterized from
Thauera aromatica
. The enzyme is fully reversible and forms exclusively the 2-(
R
)-benzylsuccinyl-CoA isomer. Only some close chemical analogs of the substrates are accepted by the enzyme: succinate was partially replaced by maleate or methylsuccinate, and (
R
)-benzylsuccinate was replaced by methylsuccinate, benzylmalonate, or phenylsuccinate. In contrast to all other known CoA-transferases, the enzyme consists of two subunits of similar amino acid sequences and similar sizes (44 and 45 kDa) in an α
2
β
2
conformation. Identity of the subunits with the products of the previously identified toluene-induced
bbsEF
genes was confirmed by determination of the exact masses via electrospray-mass spectrometry. The deduced amino acid sequences resemble those of only two other characterized CoA-transferases, oxalyl-CoA:formate CoA-transferase and (
E
)-cinnamoyl-CoA:(
R
)-phenyllactate CoA-transferase, which represent a new family of CoA-transferases. As suggested by kinetic analysis, the reaction mechanism of enzymes of this family apparently involves formation of a ternary complex between the enzyme and the two substrates.
</jats:p
Techniques for the Measurement of Molecular Species of Acyl-CoA in Plants and Microalgae
No full textThe acyl-CoA pool is pivotal in cellular metabolism. The ability to provide reliable estimates of acyl-CoA abundance and distribution between molecular species in plant tissues and microalgae is essential to our understanding of lipid metabolism and acyl exchange. Acyl-CoAs are typically found in low abundance and require specific methods for extraction, separation and detection. Here we describe methods for acyl-CoA extraction and measurement in plant tissues and microalgae, with a focus on liquid chromatography hyphenated to detection techniques including ultraviolet (UV), fluorescence and mass spectrometry (MS). We address the resolution of isobaric species and the selection of columns needed to achieve this, including the analysis of branched chain acyl-CoA thioesters. For MS analyses, we describe diagnostic ions for the identification of acyl-CoA species and how these can be used for both discovery of new species (data dependent acquisition) and routine quantitation (triple quadrupole MS with multiple reaction monitoring)
Nutritional regulation of stearoyl-CoA desaturase in the bovine mammary gland
No full text Increasing the proportion of unsaturated fatty acids (UFA) in milk is believed to be beneficial in terms of human health, thereby increasing the nutritional quality of milk. The proportion of UFA in milk is mainly dependent on the proportion of UFA in the diet, the degree of biohydrogenation of UFA in the rumen, and on activity of the stearoyl-CoA desaturase (SCD) enzyme in the mammary gland. This thesis focuses on SCD in the mammary gland of dairy cows, and how SCD can be influenced by nutrition. In the first study it was shown that supplementing the diet of dairy cows with soybean oil significantly decreases mammary SCD1 expression compared with rapeseed oil or linseed oil and this was partly reflected by the lower desaturase indices in milk. In contrast, mammary SCD5 expression was much lower (3) than that of SCD1 and was not affected by dietary plant oil supplementation. To study the changes in genome-wide expression of genes in response to dietary UFA supplementation, mammary tissue samples of the same experiment were used for micro-array analysis. It was found that 972 genes were significantly affected through UFA supplementation, indicating that large transcriptional adaptations occurred in the mammary gland when diets of dairy cows were supplemented with unprotected dietary UFA. Since biopsy of the mammary gland is an invasive and costly method which presents a risk of udder infection, the use of milk somatic cells as a non-invasive, alternative source of mRNA was investigated in the second experiment. Since there was a significant relationship between SCD1 expression in milk somatic cells and mammary tissue, it can be concluded that milk somatic cells can be used as a source of mRNA to study SCD1 expression in dairy cows. To study the effects of acetate (Ac) and β-hydroxybutyrate (BHBA) as well as various long-chain fatty acids (LCFA) on mammary SCD expression, a bovine mammary epithelial cell line (MAC-T) was used in the third experiment. This study showed that Ac up-regulates expression of SCD1 and acetyl-CoA carboxylase in MAC-T cells, which indicates that Ac may increase desaturation and de novo synthesis of fatty acids in the bovine mammary gland. In addition it was shown that expression of sterol regulatory binding protein 1 (SREBP-1) and insulin-induced gene 1 protein (INSIG-1) was related to the expression of several lipogenic genes, thereby strengthening the support for the role of SREBP-1 and INSIG-1 as central regulators of lipogenesis in the bovine mammary gland. Overall, it can be concluded that saturated LCFA have little or no effect on SCD1 expression in the bovine mammary gland, whereas unsaturated LCFA inhibit mammary SCD1 expression. The regulation of SCD1 in the bovine mammary gland by LCFA appears to be, at least partly, regulated by the transcription factors SREBP-1 and INSIG-1. Based on the in vitro research it appears that short-chain fatty acids, in particular Ac, upregulate mammary SCD1 expression, although further research is needed to verify if short-chain fatty acids induce SCD1 expression in the bovine mammary gland. The recently discovered isoform SCD5 is expressed in bovine mammary tissue, although contribution to ∆9-desaturation of fatty acids appears to be quite low. </p
- …
