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Toshiyuki Fukao - One of the best experts on this subject based on the ideXlab platform.
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metabolic encephalopathy in beta ketothiolase deficiency the first report from india
Brain & Development, 2014Co-Authors: Radha Rama Devi Akella, Yuka Aoyama, Chihiro Mori, Lokesh Lingappa, Rohit Cariappa, Toshiyuki FukaoAbstract:Abstract Beta-ketothiolase deficiency, or mitochondrial Acetoacetyl-CoA thiolase (T2) deficiency, is a rare autosomal recessive disorder affecting isoleucine catabolism and ketone body metabolism. A patient from South India presented with acute ketoacidosis at 11 months of age. During the acute crisis the C5OH (2-methyl-3-hydroxybutyryl) carnitine and C5:1 (tiglyl) carnitine were elevated and large amounts of 2-methyl-3-hydroxybutyrate, tiglylglycine, and 2-methylacetoacetate were excreted. Brain CT showed bilateral basal ganglia lesions. Potassium ion-activated Acetoacetyl-CoA thiolase activity was deficient in the patient’s fibroblasts. The patient is a homozygote for a novel c.578T>G (M193R) mutation. This is the first report of T2 deficiency confirmed by enzyme and molecular analysis from India.
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A structural mapping of mutations causing succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency
Journal of inherited metabolic disease, 2013Co-Authors: Naeem Shafqat, Toshiyuki Fukao, Kate L. Kavanagh, Jörn Oliver Sass, Ernst Christensen, Wen Hwa Lee, Udo Oppermann, Wyatt W. YueAbstract:Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare inherited metabolic disorder of ketone metabolism, characterized by ketoacidotic episodes and often permanent ketosis. To date there are ∼20 disease-associated alleles on the OXCT1 gene that encodes the mitochondrial enzyme SCOT. SCOT catalyzes the first, rate-limiting step of ketone body utilization in peripheral tissues, by transferring a CoA moiety from succinyl-CoA to form Acetoacetyl-CoA, for entry into the tricarboxylic acidcycleforenergyproduction.We have determined the crystal structure of human SCOT, provid- ing a molecular understanding of the reported mutations based on their potential structural effects. An interactive version of this manuscript (which may contain additional mutations appended after acceptance of this manuscript) may be found on the web address: http://www.thesgc.org/jimd/SCOT. Abbreviations
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differences between human and rodent pancreatic islets low pyruvate carboxylase atp citrate lyase and pyruvate carboxylation and high glucose stimulated acetoacetate in human pancreatic islets
Journal of Biological Chemistry, 2011Co-Authors: Michael J Macdonald, Noaman Hasan, Melissa J Longacre, Scott W Stoker, Mindy A Kendrick, Ansaya Thonpho, Laura J Brown, Sarawut Jitrapakdee, Toshiyuki FukaoAbstract:Anaplerosis, the net synthesis in mitochondria of citric acid cycle intermediates, and cataplerosis, their export to the cytosol, have been shown to be important for insulin secretion in rodent beta cells. However, human islets may be different. We observed that the enzyme activity, protein level, and relative mRNA level of the key anaplerotic enzyme pyruvate carboxylase (PC) were 80–90% lower in human pancreatic islets compared with islets of rats and mice and the rat insulinoma cell line INS-1 832/13. Activity and protein of ATP citrate lyase, which uses anaplerotic products in the cytosol, were 60–75% lower in human islets than in rodent islets or the cell line. In line with the lower PC, the percentage of glucose-derived pyruvate that entered mitochondrial metabolism via carboxylation in human islets was only 20–30% that in rat islets. This suggests human islets depend less on pyruvate carboxylation than rodent models that were used to establish the role of PC in insulin secretion. Human islets possessed high levels of succinyl-CoA:3-ketoacid-CoA transferase, an enzyme that forms acetoacetate in the mitochondria, and Acetoacetyl-CoA synthetase, which uses acetoacetate to form acyl-CoAs in the cytosol. Glucose-stimulated human islets released insulin similarly to rat islets but formed much more acetoacetate. β-Hydroxybutyrate augmented insulin secretion in human islets. This information supports previous data that indicate beta cells can use a pathway involving succinyl-CoA:3-ketoacid-CoA transferase and Acetoacetyl-CoA synthetase to synthesize and use acetoacetate and suggests human islets may use this pathway more than PC and citrate to form cytosolic acyl-CoAs.
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lower succinyl coa 3 ketoacid coa transferase scot and atp citrate lyase in pancreatic islets of a rat model of type 2 diabetes knockdown of scot inhibits insulin release in rat insulinoma cells
Archives of Biochemistry and Biophysics, 2010Co-Authors: Noaman Hasan, Toshiyuki Fukao, Melissa J Longacre, Mindy A Kendrick, Mohammed Seed Ahmed, Harvest F Gu, Claesgoran Ostenson, Michael J MacdonaldAbstract:Abstract Succinyl-CoA:3-ketoacid-CoA transferase (SCOT) is a mitochondrial enzyme that catalyzes the reversible transfer of coenzyme-A from Acetoacetyl-CoA to succinate to form acetoacetate and succinyl-CoA. mRNAs of SCOT and ATP citrate lyase were decreased 55% and 58% and enzyme activities were decreased >70% in pancreatic islets of the GK rat, a model of type 2 diabetes. INS-1 832/13 cells were transfected with shRNAs targeting SCOT mRNA to generate cell lines with reduced SCOT activity. Two cell lines with >70% knockdown of SCOT activity showed >70% reduction in glucose- or methyl succinate-plus-β-hydroxybutyrate-stimulated insulin release. Less inhibition of insulin release was observed with two cell lines with less knockdown of SCOT. Previous studies showed knockdown of ATP citrate lyase in INS-1 832/13 cells does not lower insulin release. The results further support work that suggests mitochondrial pathways involving SCOT which supply acetoacetate for export to the cytosol are important for insulin secretion.
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different clinical presentation in siblings with mitochondrial acetoacetyl coa thiolase deficiency and identification of two novel mutations
Tohoku Journal of Experimental Medicine, 2010Co-Authors: Susanne Thummler, Toshiyuki Fukao, Didier Dupont, Cecile Acquaviva, Dominique De RicaudAbstract:Mitochondrial Acetoacetyl-CoA thiolase (T2) catalyzes 2-methylAcetoacetyl-CoA cleavage into acetyl-CoA and propionyl-CoA in isoleucine catabolism and interconversion between acetyl-CoA and Acetoacetyl-CoA in ketone body metabolism. T2 deficiency is a rare metabolic disease of autosomal recessive inheritance. The disorder is characterized by intermittent ketoacidotic episodes. The onset of clinical symptoms is in the infant or toddler period. The frequency of episodes declines with age, stopping before adolescence. Here we report two siblings with this disorder. The proband (GK65) is a French girl born from non-consanguineous parents. She presented several ketoacidotic episodes with 5 hospitalizations from age 2 to 4 years, the first of them complicated by ketoacidotic coma. Minor episodes, which are generally provoked by infections or high protein intake, still persist at age of 16 years. Molecular analysis of the T2 gene has revealed the compound heterozygosity of c.578T>C (M193T) and IVS8+5g>t. The latter mutation results in skipping of exon 8. In contrast, the younger brother (GK65b) had a unique ketoacidotic crisis at the age of 6 years that is the oldest-age first crisis among T2-deficient patients reported thus far. Despite the mild phenotype, he carried the same T2 gene mutations as his sister (GK65). Furthermore, T2 catalytic activity and T2 protein were not detected in the fibroblasts derived from GK65 and GK65b. In conclusion, the siblings with the same T2 gene mutations present different clinical severity. Diagnostic testing for asymptomatic siblings is important in the management of T2-deficient families.
Naomi Kondo - One of the best experts on this subject based on the ideXlab platform.
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identification of an alu mediated tandem duplication of exons 8 and 9 in a patient with mitochondrial acetoacetyl coa thiolase t2 deficiency
Molecular Genetics and Metabolism, 2007Co-Authors: Toshiyuki Fukao, Gaixiu Zhang, Marieodile Rolland, Marietherese Zabot, Nathalie Guffon, Yusuke Aoki, Naomi KondoAbstract:A tandem repeat of exons 8 and 9 was identified in the cDNA for mitochondrial Acetoacetyl-CoA thiolase (T2) in a typical T2 deficient patient. Routine mutation analysis using PCR at the genomic level had failed to identify any mutations. Alu element-mediated unequal homologous recombination between an Alu-Jo in intron 7 and another Alu-Jo in intron 9 appears to be responsible for this duplication.
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crystallographic and kinetic studies of human mitochondrial acetoacetyl coa thiolase the importance of potassium and chloride ions for its structure and function
Biochemistry, 2007Co-Authors: Antti M Haapalainen, Päivi Pirilä, Gitte Merilainen, Naomi Kondo, Toshiyuki Fukao, Rikkert K WierengaAbstract:Thiolases are CoA-dependent enzymes which catalyze the formation of a carbon-carbon bond in a Claisen condensation step and its reverse reaction via a thiolytic degradation mechanism. Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase (T2) is important in the pathways for the synthesis and degradation of ketone bodies as well as for the degradation of 2-methylAcetoacetyl-CoA. Human T2 deficiency has been identified in more than 60 patients. A unique property of T2 is its activation by potassium ions. High-resolution human T2 crystal structures are reported for the apo form and the CoA complex, with and without a bound potassium ion. The potassium ion is bound near the CoA binding site and the catalytic site. Binding of the potassium ion at this low-affinity binding site causes the rigidification of a CoA binding loop and an active site loop. Unexpectedly, a high-affinity binding site for a chloride ion has also been identified. The chloride ion is copurified, and its binding site is at the dimer interface, near two catalytic loops. A unique property of T2 is its ability to use 2-methyl-branched Acetoacetyl-CoA as a substrate, whereas the other structurally characterized thiolases cannot utilize the 2-methylated compounds. The kinetic measurements show that T2 can degrade Acetoacetyl-CoA and 2-methylAcetoacetyl-CoA with similar catalytic efficiencies. For both substrates, the turnover numbers increase approximately 3-fold when the potassium ion concentration is increased from 0 to 40 mM KCl. The structural analysis of the active site of T2 indicates that the Phe325-Pro326 dipeptide near the catalytic cavity is responsible for the exclusive 2-methyl-branched substrate specificity.
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two novel mutations in mitochondrial acetoacetyl coa thiolase deficiency
Journal of Inherited Metabolic Disease, 2005Co-Authors: L Mrazova, Naomi Kondo, Toshiyuki Fukao, K Halovd, E Gregova, V Kohut, D Přibyl, Petr Chrastina, E PospisilovaAbstract:We report a new patient with Acetoacetyl-CoA thiolase deficiency in whom we found two new missense mutations.
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high resolution crystal structures of human cytosolic thiolase ct a comparison of the active sites of human ct bacterial thiolase and bacterial kas i
Journal of Molecular Biology, 2005Co-Authors: Herkko Sikkila, Naomi Kondo, Petri Kursula, Toshiyuki Fukao, Rikkert K WierengaAbstract:Thiolases belong to a superfamily of condensing enzymes that includes also β-ketoacyl acyl carrier protein synthases (KAS enzymes), involved in fatty acid synthesis. Here, we describe the high resolution structure of human cytosolic Acetoacetyl-CoA thiolase (CT), both unliganded (at 2.3 A resolution) and in complex with CoA (at 1.6 A resolution). CT catalyses the condensation of two molecules of acetyl-CoA to Acetoacetyl-CoA, which is the first reaction of the metabolic pathway leading to the synthesis of cholesterol. CT is a homotetramer of exact 222 symmetry. There is an excess of positively charged residues at the interdimer surface leading towards the CoA-binding pocket, possibly important for the efficient capture of substrates. The geometry of the catalytic site, including the three catalytic residues Cys92, His 353, Cys383, and the two oxyanion holes, is highly conserved between the human and bacterial Zoogloea ramigera thiolase. In human CT, the first oxyanion hole is formed by Wat38 (stabilised by Asn321) and NE2(His353), and the second by N(Cys92) and N(Gly385). The active site of this superfamily is constructed on top of four active site loops, near Cys92, Asn321, His353, and Cys383, respectively. These loops were used for the superpositioning of CT on the bacterial thiolase and on the Escherichia coli KAS I. This comparison indicates that the two thiolase oxyanion holes also exist in KAS I at topologically equivalent positions. Interestingly, the hydrogen bonding interactions at the first oxyanion hole are different in thiolase and KAS I. In KAS I, the hydrogen bonding partners are two histidine NE2 atoms, instead of a water and a NE2 side-chain atom in thiolase. The second oxyanion hole is in both structures shaped by corresponding main chain peptide NH-groups. The possible importance of bound water molecules at the catalytic site of thiolase for the reaction mechanism is discussed.
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mitochondrial acetoacetyl coa thiolase t2 deficiency t2 deficient patients with mild mutation s were previously misinterpreted as normal by the coupled assay with tiglyl coa
Pediatric Research, 2004Co-Authors: Gaixiu Zhang, Toshiyuki Fukao, Marieodile Rolland, Marietherese Zabot, Gilles Renom, Elias Touma, Masashi Kondo, Naoki Matsuo, Naomi KondoAbstract:Mitochondrial Acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism that affects the catabolism of isoleucine and ketone bodies. This disorder is characterized by intermittent ketoacidotic episodes. Recently, we diagnosed T2 deficiency in two patients (GK45 and GK47) by the absence of potassium ion-activated Acetoacetyl-CoA thiolase activity, whereas these patients were previously misinterpreted as normal by a coupled assay with tiglyl-CoA as a substrate. This method has been widely used for the enzymatic diagnosis of the T2 deficiency in the United States and Europe. We hypothesized that some residual T2 activity showed normal results in the assay. To prove this hypothesis, we analyzed these two patients together with three typical T2-deficient patients (GK46, GK49, and GK50) at the DNA level. Expression analysis of mutant cDNAs clearly showed that GK45 and GK47 had “mild” mutations (A132G, D339-V340insD) that retained some residual T2 activity, at least one of two mutant alleles, whereas the other three patients had null mutations (c.52–53insC, G152A, H397D, and IVS8+1g>t) in either allele. These results raise the possibility that T2-deficient patients with mild mutations have been misinterpreted as normal by the coupled assay with tiglyl-CoA.
Rikkert K Wierenga - One of the best experts on this subject based on the ideXlab platform.
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crystallographic and kinetic studies of human mitochondrial acetoacetyl coa thiolase the importance of potassium and chloride ions for its structure and function
Biochemistry, 2007Co-Authors: Antti M Haapalainen, Päivi Pirilä, Gitte Merilainen, Naomi Kondo, Toshiyuki Fukao, Rikkert K WierengaAbstract:Thiolases are CoA-dependent enzymes which catalyze the formation of a carbon-carbon bond in a Claisen condensation step and its reverse reaction via a thiolytic degradation mechanism. Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase (T2) is important in the pathways for the synthesis and degradation of ketone bodies as well as for the degradation of 2-methylAcetoacetyl-CoA. Human T2 deficiency has been identified in more than 60 patients. A unique property of T2 is its activation by potassium ions. High-resolution human T2 crystal structures are reported for the apo form and the CoA complex, with and without a bound potassium ion. The potassium ion is bound near the CoA binding site and the catalytic site. Binding of the potassium ion at this low-affinity binding site causes the rigidification of a CoA binding loop and an active site loop. Unexpectedly, a high-affinity binding site for a chloride ion has also been identified. The chloride ion is copurified, and its binding site is at the dimer interface, near two catalytic loops. A unique property of T2 is its ability to use 2-methyl-branched Acetoacetyl-CoA as a substrate, whereas the other structurally characterized thiolases cannot utilize the 2-methylated compounds. The kinetic measurements show that T2 can degrade Acetoacetyl-CoA and 2-methylAcetoacetyl-CoA with similar catalytic efficiencies. For both substrates, the turnover numbers increase approximately 3-fold when the potassium ion concentration is increased from 0 to 40 mM KCl. The structural analysis of the active site of T2 indicates that the Phe325-Pro326 dipeptide near the catalytic cavity is responsible for the exclusive 2-methyl-branched substrate specificity.
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high resolution crystal structures of human cytosolic thiolase ct a comparison of the active sites of human ct bacterial thiolase and bacterial kas i
Journal of Molecular Biology, 2005Co-Authors: Herkko Sikkila, Naomi Kondo, Petri Kursula, Toshiyuki Fukao, Rikkert K WierengaAbstract:Thiolases belong to a superfamily of condensing enzymes that includes also β-ketoacyl acyl carrier protein synthases (KAS enzymes), involved in fatty acid synthesis. Here, we describe the high resolution structure of human cytosolic Acetoacetyl-CoA thiolase (CT), both unliganded (at 2.3 A resolution) and in complex with CoA (at 1.6 A resolution). CT catalyses the condensation of two molecules of acetyl-CoA to Acetoacetyl-CoA, which is the first reaction of the metabolic pathway leading to the synthesis of cholesterol. CT is a homotetramer of exact 222 symmetry. There is an excess of positively charged residues at the interdimer surface leading towards the CoA-binding pocket, possibly important for the efficient capture of substrates. The geometry of the catalytic site, including the three catalytic residues Cys92, His 353, Cys383, and the two oxyanion holes, is highly conserved between the human and bacterial Zoogloea ramigera thiolase. In human CT, the first oxyanion hole is formed by Wat38 (stabilised by Asn321) and NE2(His353), and the second by N(Cys92) and N(Gly385). The active site of this superfamily is constructed on top of four active site loops, near Cys92, Asn321, His353, and Cys383, respectively. These loops were used for the superpositioning of CT on the bacterial thiolase and on the Escherichia coli KAS I. This comparison indicates that the two thiolase oxyanion holes also exist in KAS I at topologically equivalent positions. Interestingly, the hydrogen bonding interactions at the first oxyanion hole are different in thiolase and KAS I. In KAS I, the hydrogen bonding partners are two histidine NE2 atoms, instead of a water and a NE2 side-chain atom in thiolase. The second oxyanion hole is in both structures shaped by corresponding main chain peptide NH-groups. The possible importance of bound water molecules at the catalytic site of thiolase for the reaction mechanism is discussed.
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the catalytic cycle of biosynthetic thiolase a conformational journey of an acetyl group through four binding modes and two oxyanion holes
Biochemistry, 2002Co-Authors: Petri Kursula, Juha Ojala, And Annemarie Lambeir, Rikkert K WierengaAbstract:Biosynthetic thiolase catalyzes the formation of Acetoacetyl-CoA from two molecules of acetyl-CoA. This is a key step in the synthesis of many biological compounds, including steroid hormones and ketone bodies. The thiolase reaction involves two chemically distinct steps; during acyl transfer, an acetyl group is transferred from acetyl-CoA to Cys89, and in the Claisen condensation step, this acetyl group is further transferred to a second molecule of acetyl-CoA, generating Acetoacetyl-CoA. Here, new crystallographic data for Zoogloea ramigera biosynthetic thiolase are presented, covering all intermediates of the thiolase catalytic cycle. The high-resolution structures indicate that the acetyl group goes through four conformations while being transferred from acetyl-CoA via the acetylated enzyme to Acetoacetyl-CoA. This transfer is catalyzed in a rigid cavity lined by mostly hydrophobic side chains, in addition to the catalytic residues Cys89, His348, and Cys378. The structures highlight the importance of ...
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The catalytic cycle of biosynthetic thiolase: a conformational journey of an acetyl group through four binding modes and two oxyanion holes.
Biochemistry, 2002Co-Authors: Petri Kursula, Juha Ojala, ‖ And Anne-marie Lambeir, Rikkert K WierengaAbstract:Biosynthetic thiolase catalyzes the formation of Acetoacetyl-CoA from two molecules of acetyl-CoA. This is a key step in the synthesis of many biological compounds, including steroid hormones and ketone bodies. The thiolase reaction involves two chemically distinct steps; during acyl transfer, an acetyl group is transferred from acetyl-CoA to Cys89, and in the Claisen condensation step, this acetyl group is further transferred to a second molecule of acetyl-CoA, generating Acetoacetyl-CoA. Here, new crystallographic data for Zoogloea ramigera biosynthetic thiolase are presented, covering all intermediates of the thiolase catalytic cycle. The high-resolution structures indicate that the acetyl group goes through four conformations while being transferred from acetyl-CoA via the acetylated enzyme to Acetoacetyl-CoA. This transfer is catalyzed in a rigid cavity lined by mostly hydrophobic side chains, in addition to the catalytic residues Cys89, His348, and Cys378. The structures highlight the importance of an oxyanion hole formed by a water molecule and His348 in stabilizing the negative charge on the thioester oxygen atom of acetyl-CoA at two different steps of the reaction cycle. Another oxyanion hole, composed of the main chain nitrogen atoms of Cys89 and Gly380, complements a negative charge of the thioester oxygen anion of the acetylated intermediate, stabilizing the tetrahedral transition state of the Claisen condensation step. The reactivity of the active site may be modulated by hydrogen bonding networks extending from the active site toward the back of the molecule.
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a biosynthetic thiolase in complex with a reaction intermediate the crystal structure provides new insights into the catalytic mechanism
Structure, 1999Co-Authors: Yorgo Modis, Rikkert K WierengaAbstract:Abstract Background: Thiolases are ubiquitous and form a large family of dimeric or tetrameric enzymes with a conserved, five-layered α β α β α catalytic domain. Thiolases can function either degradatively, in the β -oxidation pathway of fatty acids, or biosynthetically. Biosynthetic thiolases catalyze the biological Claisen condensation of two molecules of acetyl-CoA to form Acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in a wide range of biosynthetic pathways, including those that generate cholesterol, steroid hormones, and various energy-storage molecules. Results: The crystal structure of the tetrameric biosynthetic thiolase from Zoogloea ramigera has been determined at 2.0 A resolution. The structure contains a striking and novel ‘cage-like' tetramerization motif, which allows for some hinge motion of the two tight dimers with respect to each other. The protein crystals were flash-frozen after a short soak with the enzyme's substrate, Acetoacetyl-CoA. A reaction intermediate was thus trapped: the enzyme tetramer is acetylated at Cys89 and has a CoA molecule bound in each of its active-site pockets. Conclusions: The shape of the substrate-binding pocket reveals the basis for the short-chain substrate specificity of the enzyme. The active-site architecture, and in particular the position of the covalently attached acetyl group, allow a more detailed reaction mechanism to be proposed in which Cys378 is involved in both steps of the reaction. The structure also suggests an important role for the thioester oxygen atom of the acetylated enzyme in catalysis.
Alexander Steinbuchel - One of the best experts on this subject based on the ideXlab platform.
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synthesis of poly 3 hydroxybutyrate co 3 hydroxyvalerate from unrelated carbon sources in engineered rhodospirillum rubrum
Fems Microbiology Letters, 2015Co-Authors: Daniel Heinrich, Alexander Steinbuchel, Matthias RabergAbstract:Different genes encoding pyridine nucleotide transhydrogenases ( pntAB, udhA ) and Acetoacetyl-CoA reductases ( phaB ) were heterologously overexpressed in Rhodospirillum rubrum S1. A recombinant strain, which harbored the gene encoding the membrane-bound transhydrogenase PntAB from Escherichia coli MG1655 and the phaB1 gene coding for an NADPH-dependent Acetoacetyl-CoA reductase from Ralstonia eutropha H16, accumulated poly(3-hydroxybutyrate- co -3-hydroxyvalerate) [Poly(3HB- co -3HV)] with a 3HV fraction of up to 13 mol% from fructose. This was a 13-fold increase of the 3HV content when compared to the wild-type strain. Higher contents of 3HV are known to reduce the brittleness of this polymer, which is advantageous for most applications. The engineered R. rubrum strain was also able to synthesize this industrially relevant copolymer from CO2 and CO from artificial synthesis gas (syngas) with a 3HV content of 56 mol%. The increased incorporation of 3HV was attributed to an excess of propionyl-CoA, which was generated from threonine and related amino acids to compensate for the intracellular redox imbalance resulting from the transhydrogenase reaction. Thereby, our study presents a novel, molecular approach to alter the composition of bacterial PHAs independently from external precursor supply. Moreover, this study also provides a promising production strain for syngas-derived second-generation biopolymers.
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s 3 hydroxyacyl coa dehydrogenase enoyl coa hydratase fadb from fatty acid degradation operon of ralstonia eutropha h16
AMB Express, 2014Co-Authors: Elena Volodina, Alexander SteinbuchelAbstract:In this study (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (H16_A0461/FadB’, gene ID: 4247876) from one of two active fatty acid degradation operons of Ralstonia eutropha H16 has been heterologously expressed in Escherichia coli, purified as protein possessing a His-Tag and initially characterized. FadB’ is an enzyme with two catalytic domains exhibiting a single monomeric structure and possessing a molecular weight of 86 kDa. The C-terminal part of the enzyme harbors enoyl-CoA hydratase activity and is able to convert trans-crotonyl-CoA to 3-hydroxybutyryl-CoA. The N-terminal part of FadB’ comprises an NAD+ binding site and is responsible for 3-hydroxyacyl-CoA dehydrogenase activity converting (S)-3-hydroxybutyryl-CoA to Acetoacetyl-CoA. Enoyl-CoA hydratase activity was detected spectrophotometrically with trans-crotonyl-CoA. (S)-3-Hydroxyacyl-CoA dehydrogenase activity was measured in both directions with Acetoacetyl-CoA and 3-hydroxybutyryl-CoA. FadB’ was found to be strictly stereospecific to (S)-3-hydroxybutyryl-CoA and to prefer NAD+. The K m value for Acetoacetyl-CoA was 48 μM and V max 149 μmol mg−1 min−1. NADP(H) was utilized at a rate of less than 10% in comparison to activity with NAD(H). FadB’ exhibited optimal activity at pH 6–7 and the activity decreased at alkaline and acidic pH values. Acetyl-CoA, propionyl-CoA and CoA were found to have an inhibitory effect on FadB’. This study is a first report on biochemical properties of purified (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase with the inverted domain order from R. eutropha H16. In addition to fundamental information about FadB’ and fatty acid metabolism, FadB’ might be also interesting for biotechnological applications.
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Central metabolism of R. eutropha H16 and mutant PHB-4 with regard to the results of proteome analyses.
2014Co-Authors: Matthias Raberg, Birgit Voigt, Michael Hecker, Alexander SteinbuchelAbstract:The numbers in the scheme indicate the following involved enzymes: 1, glucokinase; 2, phosphogluconate dehydratase; 3, phospho-2-keto-3-desoxygluconate aldolase; 4, glyceraldehyde-3-phosphate dehydrogenase; 5, phosphoglycerate dehydrogenase; 6, phosphoglyceromutase; 7, enolase; 8, pyruvate kinase; 9, pyruvate dehydrogenase/decarboxylase (E1 of PDHC); 10, dihydrolipoamide acetyltransferase (E1 of PDHC); 11, dihydrolipoamide dehydrogenase (E3 of PDHC); 12, acetoin dehydrogenase enzyme system; 13, acetyl-CoA acetyltransferase; 14, Acetoacetyl-CoA reductase; 15, PHB synthase; 16, 3-oxoacid-CoA transferase; 17, 3-hydroxybutyrate dehydrogenase; 18, citrate synthase; 19, aconitase; 20, isocitrate dehydrogenase; 21, 2-oxoacid dehydrogenase multienzyme complex; 22, succinyl-CoA synthetase; 23, succinate dehydrogenase; 24, fumarase; 25, malate dehydrogenase; 26, citrate lyase.
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expression of polyhydroxyalkanoic acid biosynthesis genes in methylotrophic bacteria relying on the ribulose monophosphate pathway
Applied Microbiology and Biotechnology, 1993Co-Authors: Christina Follner, Wolfgang Babel, Henry E. Valentin, Alexander SteinbuchelAbstract:Four representatives of methylotrophic bacteria relying on the ribulose monophosphate (RMP) pathway were investigated for their capability to synthesize polyhydroxyalkanoic acids (PHA). In Methylophilus methylotrophus B115, Methylobacillus glycogenes strains B121 and B53 and Acetobacter methanolicus B58 no \-ketothiolase, acetoacetyl-coenzyme A (CoA) reductase or PHA synthase could be detected, and hybridization experiments using heterologous DNA probes derived from PHA-biosynthesis genes of Methylobacterium extorquens or Alcaligenes eutrophus gave no evidence for the presence of the corresponding genes in these PHA-negative methylotrophic bacteria. Fragments harbouring a cluster of PHA-biosynthesis genes of A. eutrophus or Chromatium vinosum or isolated PHA synthase structural genes of M. extorquens, Rhodospirillum rubrum or Rhodobacter sphaeroides were mobilized into the RMP pathway bacteria mentioned above. Only transconjugants, which harboured the PHA-biosynthesis genes of A. eutrophus or C. vinosum, expressed active \-ketothiolase, Acetoacetyl-CoA reductase and PHA synthase and accumulated poly(3-hydroxybutyric acid) (PHB). Highest amounts of PHB (up to 15% of the cellular dry weight) were accumulated in transconjugants of Methylophilus methylotrophus B115 or of Methylobacillus glycogenes strains B121 and B53 harbouring the PHA-biosynthesis genes of C. vinosum.
Sarah Shefer - One of the best experts on this subject based on the ideXlab platform.
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regulation of early cholesterol biosynthesis in rat liver effects of sterols bile acids lovastatin and bm 15 766 on 3 hydroxy 3 methylglutaryl coenzyme a synthase and acetoacetyl coenzyme a thiolase activities
Hepatology, 1998Co-Authors: Akira Honda, Ashok K. Batta, Guorong Xu, Stephen G Tint, Sarah SheferAbstract:Cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase catalyzes the formation of HMG-CoA, the substrate for the rate-controlling enzyme in the cholesterol biosynthetic pathway. To explore the regulation in liver, we developed a new, accurate, and reliable reversed-phase ion-pair chromatographic assay that uses nonradioactive substrates and n-propionyl coenzyme A as an internal recovery standard. The hepatic activities were measured in rats treated with cholesterol, sitosterol, cholic acid, deoxycholic acid, ursodeoxycholic acid, cholestyramine, bile fistula, lovastatin, and BM 15.766, an inhibitor of 7-dehydrocholesterol Δ7-reductase, and were compared with microsomal HMG-CoA reductase and cytosolic acetoacetyl coenzyme A (AcAc-CoA) thiolase activities. HMG-CoA synthase activity was effectively suppressed in synchrony with HMG-CoA reductase activity by treatments with cholesterol (−41%, P< .05), cholic acid (−72%, P< .005), and deoxycholic acid (−62%, P< .05). However, ursodeoxycholic acid increased activity 84% (P< .05) and intravenous sitosterol did not change activity. AcAc-CoA thiolase activities also paralleled HMG-CoA reductase and HMG-CoA synthase activities, but differences were not statistically significant. In contrast to inhibition, up-regulation of hepatic HMG-CoA synthase activities by cholestyramine, bile fistula, and lovastatin was much less than HMG-CoA reductase activities. In addition, BM 15.766 did not stimulate synthase activity, whereas lovastatin increased activity 2.4-fold. Thus, hepatic HMG-CoA synthase activity was regulated coordinately with HMG-CoA reductase, and responded more forcefully to regulatory stimuli than Acetoacetyl-CoA thiolase activity but usually less than HMG-CoA reductase.
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down regulation of cholesterol biosynthesis in sitosterolemia diminished activities of acetoacetyl coa thiolase 3 hydroxy 3 methylglutaryl coa synthase reductase squalene synthase and 7 dehydrocholesterol delta7 reductase in liver and mononuclear leu
Journal of Lipid Research, 1998Co-Authors: Akira Honda, G. Stephen Tint, Lien B Nguyen, Ashok K. Batta, Sarah SheferAbstract:Sitosterolemia is a recessively inherited disorder characterized by abnormally increased plasma and tissue plant sterol concentrations. Patients have markedly reduced whole body cholesterol biosynthesis associated with sup- pressed hepatic, ileal, and mononuclear leukocyte 3-hydroxy- 3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate- controlling enzyme in cholesterol biosynthetic pathway, coupled with significantly increased low density lipoprotein (LDL) receptor expression. To investigate the mechanism of down- regulated cholesterol biosynthesis, we assayed several other key enzymes in the cholesterol biosynthetic pathway includ- ing Acetoacetyl-CoA thiolase, HMG-CoA synthase, squalene synthase, and 7-dehydrocholesterol D 7 -reductase activities in liver and freshly isolated mononuclear leukocytes from four sitosterolemic patients and 19 controls. Hepatic acetoacetyl- CoA thiolase, HMG-CoA synthase, reductase, and squalene synthase activities were significantly decreased ( P , 0.05) 2 39%, 2 54%, 2 76%, and 2 57%, respectively, and 7-dehydro- cholesterol D 7 -reductase activity tended to be lower ( 2 35%) in the sitosterolemic compared with control subjects. The re- duced HMG-CoA synthase, reductase, and squalene synthase activities were also found in mononuclear leukocytes from a sitosterolemic patient. Thus, reduced cholesterol synthesis is caused not only by decreased HMG-CoA reductase but also by the coordinate down-regulation of entire pathway of choles- terol biosynthesis. These results suggest that inadequate cholesterol production in sitosterolemia is due to abnormal down-regulation of early, intermediate, and late enzymes in the cholesterol biosynthetic pathway rather than a single in- herited defect in the HMG-CoA reductase gene.— Honda, A., G. Salen, L. B. Nguyen, G. S. Tint, A. K. Batta, and S. Shefer. Down-regulation of cholesterol biosynthesis in sitosterolemia: diminished activities of Acetoacetyl-CoA thiolase, 3-hydroxy-3- methylglutaryl-CoA synthase, reductase, squalene synthase, and 7-dehydrocholesterol D 7 -reductase in liver and mononu- clear leukocytes. J. Lipid Res. 1998. 39: 44-50.
