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Georg Fuchs - One of the best experts on this subject based on the ideXlab platform.
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study of an alternate glyoxylate cycle for acetate assimilation by rhodobacter sphaeroides
Molecular Microbiology, 2006Co-Authors: Birgit E Alber, Regina Spanheimer, Christa Ebenaujehle, Georg FuchsAbstract:Organisms, which grow on organic substrates that are metabolized via acetyl-CoA, are faced with the problem to form all cell constituents from this C(2)-unit. The problem was solved by the seminal work of Kornberg and is known as the glyoxylate cycle. However, many bacteria are known to not contain isocitrate lyase, the key enzyme of this pathway. This problem was addressed in acetate-grown Rhodobacter sphaeroides. An acetate-minus mutant identified by transposon mutagenesis was affected in the gene for beta-ketothiolase forming acetoacetyl-CoA from two molecules of acetyl-CoA. This enzyme activity was missing in this mutant, which grew on acetoacetate and on acetate plus glyoxylate. A second acetate/acetoacetate-minus mutant was affected in the gene for a putative mesaconyl-CoA hydratase, an enzyme which catalyses the hydration of mesaconyl-CoA to beta-methylmalyl-CoA. Beta-methylmalyl-CoA is further cleaved into glyoxylate and Propionyl-CoA. These results as well as identification of acetate-upregulated proteins by two-dimensional gel electrophoresis lead to the proposal of a new pathway for acetate assimilation. In a first part, affected by the mutations, two molecules of acetyl-CoA and one molecule CO(2) are converted via acetoacetyl-CoA and mesaconyl-CoA to glyoxylate and Propionyl-CoA. In a second part glyoxylate and Propionyl-CoA are converted with another molecule of acetyl-CoA and CO(2) to l-malyl-CoA and succinyl-CoA.
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Characterization of acetyl-CoA/Propionyl-CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation.
European journal of biochemistry, 2003Co-Authors: Michael Hügler, Robert Krieger, Martina Jahn, Georg FuchsAbstract:Autotrophic Archaea of the family Sulfolobaceae (Crenarchaeota) use a modified 3-hydroxypropionate cycle for carbon dioxide assimilation. In this cycle the ATP-dependent carboxylations of acetyl-CoA and Propionyl-CoA to malonyl-CoA and methylmalonyl-CoA, respectively, represent the key CO2 fixation reactions. These reactions were studied in the thermophilic and acidophilic Metallosphaera sedula and are shown to be catalyzed by one single large enzyme, which acts equally well on acetyl-CoA and Propionyl-CoA. The carboxylase was purified and characterized and the genes were cloned and sequenced. In contrast to the carboxylase of most other organisms, acetyl-CoA/Propionyl-CoA carboxylase from M. sedula is active at 75 degrees C and is isolated as a stabile functional protein complex of 560 +/- 50 kDa. The enzyme consists of two large subunits of 57 kDa each representing biotin carboxylase (alpha) and carboxytransferase (gamma), respectively, and a small 18.6 kDa biotin carrier protein (beta). These subunits probably form an (alpha beta gamma)4 holoenzyme. It has a catalytic number of 28 s-1 at 65 degrees C and at the optimal pH of 7.5. The apparent Km values were 0.06 mm for acetyl-CoA, 0.07 mm for Propionyl-CoA, 0.04 mm for ATP and 0.3 mm for bicarbonate. Acetyl-CoA/Propionyl-CoA carboxylase is considered the main CO2 fixation enzyme of autotrophic members of Sulfolobaceae and the sequenced genomes of these Archaea contain the respective genes. Due to its stability the archaeal carboxylase may prove an ideal subject for further structural studies.
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propionyl coenzyme a synthase from chloroflexus aurantiacus a key enzyme of the 3 hydroxypropionate cycle for autotrophic co2 fixation
Journal of Biological Chemistry, 2002Co-Authors: Birgit E Alber, Georg FuchsAbstract:Abstract The 3-hydroxypropionate cycle has been proposed as a new autotrophic CO2 fixation pathway for the phototrophic green non-sulfur eubacterium Chloroflexus aurantiacus and for some chemotrophic archaebacteria. The cycle requires the reductive conversion of the characteristic intermediate 3-hydroxypropionate to Propionyl-CoA. The specific activity of the 3-hydroxypropionate-, CoA-, K+-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.09 μmol min−1 mg−1 protein, which was 2-fold down-regulated in heterotrophically grown cells. Unexpectedly, a single enzyme catalyzes the entire reaction sequence: 3-hydroxypropionate + MgATP + CoA + NADPH + H+ → Propionyl-CoA + MgAMP + PPi + NADP+ + H2O. The enzyme was purified 30-fold to near homogeneity and has a very large native molecular mass between 500 and 800 kDa, with subunits of about 185 kDa as judged by SDS-PAGE, suggesting a homotrimeric or homotetrameric structure. Upon incubation of this new enzyme, termed Propionyl-CoA synthase, with the proteinase trypsin, the NADPH oxidation function of the enzyme was lost, whereas the enzyme still activated 3-hydroxypropionate to its CoA-thioester and dehydrated it to acrylyl-CoA. SDS-PAGE revealed that the subunits of Propionyl-CoA synthase had been cleaved once and the N-terminal amino acid sequences of the two trypsin digestion products were determined. Two parts of the gene encoding Propionyl-CoA synthase (pcs) were identified on two contigs of an incomplete genome data base of C. aurantiacus, and the sequence of the pcs gene was completed. Propionyl-CoA synthase is a natural fusion protein of 201 kDa consisting of a CoA ligase, an enoyl-CoA hydratase, and an enoyl-CoA reductase, the reductase domain containing the trypsin cleavage site. Similar polyfunctional large enzymes are common in secondary metabolism (e.g. polyketide synthases) but rare in primary metabolism (e.g. eukaryotic type I fatty acid synthase). These results lend strong support to the operation of the proposed pathway in autotrophic CO2 fixation.
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Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation
Journal of bacteriology, 1999Co-Authors: Cástor Menéndez, Zsuzsa Bauer, Harald Huber, Nasser Gad'on, Karl-otto Stetter, Georg FuchsAbstract:The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and Propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via Propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO2 or [14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and Propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and Propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and Propionyl-CoA carboxylase are proposed to be the main CO2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.
Leonard Katz - One of the best experts on this subject based on the ideXlab platform.
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Engineered Production of Short-Chain Acyl-Coenzyme A Esters in Saccharomyces cerevisiae
ACS Synthetic Biology, 2018Co-Authors: Nicolas Krink-koutsoubelis, Anne C. Loechner, Charles M. Denby, Bastian Vögeli, Tadas Jakočiu̅nas, Anna Lechner, Hannes Link, Satoshi Yuzawa, Leonard KatzAbstract:Short-chain acyl-coenzyme A esters serve as intermediate compounds in fatty acid biosynthesis, and the production of polyketides, biopolymers and other value-added chemicals. S. cerevisiae is a model organism that has been utilized for the biosynthesis of such biologically and economically valuable compounds. However, its limited repertoire of short-chain acyl-CoAs effectively prevents its application as a production host for a plethora of natural products. Therefore, we introduced biosynthetic metabolic pathways to five different acyl-CoA esters into S. cerevisiae. Our engineered strains provide the following acyl-CoAs: Propionyl-CoA, methylmalonyl-CoA, n-butyryl-CoA, isovaleryl-CoA and n-hexanoyl-CoA. We established a yeast-specific metabolite extraction protocol to determine the intracellular acyl-CoA concentrations in the engineered strains. Propionyl-CoA was produced at 4–9 μM; methylmalonyl-CoA at 0.5 μM; and isovaleryl-CoA, n-butyryl-CoA, and n-hexanoyl-CoA at 6 μM each. The acyl-CoAs produced in t...
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Erythromycin production in Saccharopolyspora erythraea does not require a functional Propionyl-CoA carboxylase.
Molecular microbiology, 1996Co-Authors: Stefano Donadio, Michael J. Staver, Leonard KatzAbstract:Using an oligonucleotide corresponding to the consensus sequence for the biotin-binding motif, two unlinked genetic loci, bpl1 and bpl2, were cloned from the erythromycin producer Saccharopolyspora erythraea and the nucleotide sequences of a c. 4 kb segment from each determined. The two loci share a virtually identical segment of 1746 nucleotides, coinciding with most of the genes designated bcpA1 and bcpA2 present in bpl1 and bpl2, respectively. The deduced sequences of these genes are highly similar to that of the α-chain of mammalian Propionyl-CoA carboxylase. Upstream of bcpA2 lies pccB, the gene encoding the β-chain of this enzyme. Mutant strains carrying frameshift mutations in bcpA1 and pccB were constructed, but we failed to isolate insertional mutants in bcpA2. Propionyl-CoA carboxylase activity was undetectable in the pccB mutant, but was unaffected in the bcpA1-defective strain. These results indicate that pccB encodes the β-chain of Propionyl-CoA carboxylases, and suggest that the α-chain of this enzyme, which is likely to be encoded by bcpA2, is shared with some other essential biotin-dependent enzyme. The pccB mutation had no impact on erythromycin production in complex medium.
Marina A Schwab - One of the best experts on this subject based on the ideXlab platform.
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secondary mitochondrial dysfunction in propionic aciduria a pathogenic role for endogenous mitochondrial toxins
Biochemical Journal, 2006Co-Authors: Marina A Schwab, Sven W Sauer, Ullrich Brandt, Stefan Dröse, Leo G J Nijtmans, Lambertus P Van Den Heuvel, Juergen Guenther Okun, Richard J. Rodenburg, Georg F Hoffmann, Henk J. Ter LaakAbstract:Mitochondrial dysfunction during acute metabolic crises is considered an important pathomechanism in inherited disorders of propionate metabolism, i.e. propionic and methylmalonic acidurias. Biochemically, these disorders are characterized by accumulation of Propionyl-CoA and metabolites of alternative propionate oxidation. In the present study, we demonstrate uncompetitive inhibition of PDHc (pyruvate dehydrogenase complex) by Propionyl-CoA in purified porcine enzyme and in submitochondrial particles from bovine heart being in the same range as the inhibition induced by acetyl-CoA, the physiological product and known inhibitor of PDHc. Evaluation of similar monocarboxylic CoA esters showed a chain-length specificity for PDHc inhibition. In contrast with CoA esters, non-esterified fatty acidsdidnotinhibitPDHcactivity.InadditiontoPDHcinhibition, analysis ofrespiratory chain and tricarboxylic acid cycle enzymes alsorevealedaninhibitionbyPropionyl-CoAonrespiratorychain complex III and α-ketoglutarate dehydrogenase complex. To test whether impairment of mitochondrial energy metabolism is involved in the pathogenesis of propionic aciduria, we performed a thorough bioenergetic analysis in muscle biopsy specimens of two patients. In line with the in vitro results, oxidative phosphorylation was severely compromised in both patients. Furthermore, expression of respiratory chain complexes I‐IV and the amount of mitochondrial DNA were strongly decreased, and ultrastructural mitochondrial abnormalities were found, highlighting severe mitochondrial dysfunction. In conclusion, our results favour the hypothesis that toxic metabolites, in particular Propionyl-CoA, are involved in the pathogenesis of inherited disorders of propionate metabolism, sharing mechanistic similarities with propionate toxicity in micro-organisms.
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Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins
Biochemical Journal, 2006Co-Authors: Marina A Schwab, Sven W Sauer, Ullrich Brandt, Stefan Dröse, Leo G J Nijtmans, Juergen Guenther Okun, Richard J. Rodenburg, Georg F Hoffmann, Lambert P. Van Den Heuvel, Henk Ter LaakAbstract:Mitochondrial dysfunction during acute metabolic crises is considered an important pathomechanism in inherited disorders of propionate metabolism, i.e. propionic and methylmalonic acidurias. Biochemically, these disorders are characterized by accumulation of propionyl-Coenzyme A (CoA) and metabolites of alternative propionate oxidation. Here we demonstrate uncompetitive inhibition of pyruvate dehydrogenase complex (PDHc) by Propionyl-CoA in purified porcine enzyme and in submitochondrial particles from bovine heart being in the same range as the inhibition induced by acetyl-CoA, the physiological product and known inhibitor of PDHc. Evaluation of homologous monocarboxylic CoA esters showed a chain-length specificity for PDHc inhibition. In contrast to CoA esters, free fatty acids did not inhibit PDHc activity. In addition to PDHc inhibition, analysis of respiratory chain and tricarboxylic acid enzymes also revealed an inhibition of Propionyl-CoA on respiratory chain complex III and {alpha}-ketoglutarate dehydrogenase complex. To test whether impairment of mitochondrial energy metabolism is involved in the pathogenesis of propionic aciduria, we performed a thorough bioenergetic analysis in muscle biopsy specimens of two patients. In line with the in vitro results, oxidative phosphorylation was severely compromised in both patients. Furthermore, expression of respiratory chain complexes I-IV, and the amount of mitochondrial DNA were strongly decreased, and ultrastructural mitochondrial abnormalities were found, highlighting severe mitochondrial dysfunction. In conclusion, our results favour the hypothesis that toxic metabolites, in particular Propionyl-CoA, are involved in the pathogenesis of inherited disorders of propionate metabolism, sharing mechanistic similarities with propionate toxicity in microorganisms.
Henk J. Ter Laak - One of the best experts on this subject based on the ideXlab platform.
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secondary mitochondrial dysfunction in propionic aciduria a pathogenic role for endogenous mitochondrial toxins
Biochemical Journal, 2006Co-Authors: Marina A Schwab, Sven W Sauer, Ullrich Brandt, Stefan Dröse, Leo G J Nijtmans, Lambertus P Van Den Heuvel, Juergen Guenther Okun, Richard J. Rodenburg, Georg F Hoffmann, Henk J. Ter LaakAbstract:Mitochondrial dysfunction during acute metabolic crises is considered an important pathomechanism in inherited disorders of propionate metabolism, i.e. propionic and methylmalonic acidurias. Biochemically, these disorders are characterized by accumulation of Propionyl-CoA and metabolites of alternative propionate oxidation. In the present study, we demonstrate uncompetitive inhibition of PDHc (pyruvate dehydrogenase complex) by Propionyl-CoA in purified porcine enzyme and in submitochondrial particles from bovine heart being in the same range as the inhibition induced by acetyl-CoA, the physiological product and known inhibitor of PDHc. Evaluation of similar monocarboxylic CoA esters showed a chain-length specificity for PDHc inhibition. In contrast with CoA esters, non-esterified fatty acidsdidnotinhibitPDHcactivity.InadditiontoPDHcinhibition, analysis ofrespiratory chain and tricarboxylic acid cycle enzymes alsorevealedaninhibitionbyPropionyl-CoAonrespiratorychain complex III and α-ketoglutarate dehydrogenase complex. To test whether impairment of mitochondrial energy metabolism is involved in the pathogenesis of propionic aciduria, we performed a thorough bioenergetic analysis in muscle biopsy specimens of two patients. In line with the in vitro results, oxidative phosphorylation was severely compromised in both patients. Furthermore, expression of respiratory chain complexes I‐IV and the amount of mitochondrial DNA were strongly decreased, and ultrastructural mitochondrial abnormalities were found, highlighting severe mitochondrial dysfunction. In conclusion, our results favour the hypothesis that toxic metabolites, in particular Propionyl-CoA, are involved in the pathogenesis of inherited disorders of propionate metabolism, sharing mechanistic similarities with propionate toxicity in micro-organisms.
Henk Ter Laak - One of the best experts on this subject based on the ideXlab platform.
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Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins
Biochemical Journal, 2006Co-Authors: Marina A Schwab, Sven W Sauer, Ullrich Brandt, Stefan Dröse, Leo G J Nijtmans, Juergen Guenther Okun, Richard J. Rodenburg, Georg F Hoffmann, Lambert P. Van Den Heuvel, Henk Ter LaakAbstract:Mitochondrial dysfunction during acute metabolic crises is considered an important pathomechanism in inherited disorders of propionate metabolism, i.e. propionic and methylmalonic acidurias. Biochemically, these disorders are characterized by accumulation of propionyl-Coenzyme A (CoA) and metabolites of alternative propionate oxidation. Here we demonstrate uncompetitive inhibition of pyruvate dehydrogenase complex (PDHc) by Propionyl-CoA in purified porcine enzyme and in submitochondrial particles from bovine heart being in the same range as the inhibition induced by acetyl-CoA, the physiological product and known inhibitor of PDHc. Evaluation of homologous monocarboxylic CoA esters showed a chain-length specificity for PDHc inhibition. In contrast to CoA esters, free fatty acids did not inhibit PDHc activity. In addition to PDHc inhibition, analysis of respiratory chain and tricarboxylic acid enzymes also revealed an inhibition of Propionyl-CoA on respiratory chain complex III and {alpha}-ketoglutarate dehydrogenase complex. To test whether impairment of mitochondrial energy metabolism is involved in the pathogenesis of propionic aciduria, we performed a thorough bioenergetic analysis in muscle biopsy specimens of two patients. In line with the in vitro results, oxidative phosphorylation was severely compromised in both patients. Furthermore, expression of respiratory chain complexes I-IV, and the amount of mitochondrial DNA were strongly decreased, and ultrastructural mitochondrial abnormalities were found, highlighting severe mitochondrial dysfunction. In conclusion, our results favour the hypothesis that toxic metabolites, in particular Propionyl-CoA, are involved in the pathogenesis of inherited disorders of propionate metabolism, sharing mechanistic similarities with propionate toxicity in microorganisms.
