The Experts below are selected from a list of 2730 Experts worldwide ranked by ideXlab platform
Michael J. Danson - One of the best experts on this subject based on the ideXlab platform.
-
Why are the 2-Oxoacid dehydrogenase complexes so large? Generation of an active trimeric complex
Biochemical Journal, 2014Co-Authors: Nia L. Marrott, David W. Hough, Jacqueline J. T. Marshall, Dmitri I. Svergun, Susan J. Crennell, Jean M. H. Van Den Elsen, Michael J. DansonAbstract:The four-component polypeptides of the 2-Oxoacid dehydrogenase complex from the thermophilic archaeon Thermoplasma acidophilum assemble to give an active multienzyme complex possessing activity with the branched-chain 2-Oxoacids derived from leucine, isoleucine and valine, and with pyruvate. The dihydrolipoyl acyl-transferase (E2) core of the complex is composed of identical trimer-forming units that assemble into a novel 42-mer structure comprising octahedral and icosahedral geometric aspects. From our previously determined structure of this catalytic core, the inter-trimer interactions involve a tyrosine residue near the C-terminus secured in a hydrophobic pocket of an adjacent trimer like a ball-and-socket joint. In the present study, we have deleted the five C-terminal amino acids of the E2 polypeptide (IIYEI) and shown by equilibrium centrifugation that it now only assembles into a trimeric enzyme. This was confirmed by SAXS analysis, although this technique showed the presence of approximately 20% hexamers. The crystal structure of the trimeric truncated E2 core has been determined and shown to be virtually identical with the ones observed in the 42-mer, demonstrating that removal of the C-terminal anchor does not significantly affect the individual monomer or trimer structures. The truncated E2 is still able to bind both 2-Oxoacid decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components to give an active complex with catalytic activity similar to the native multienzyme complex. This is the first report of an active mini-complex for this enzyme, and raises the question of why all 2-Oxoacid dehydrogenase complexes assemble into such large structures.
-
Discovery of a putative acetoin dehydrogenase complex in the hyperthermophilic archaeon Sulfolobus solfataricus.
FEBS Letters, 2010Co-Authors: Karl A. P. Payne, David W. Hough, Michael J. DansonAbstract:Like many other aerobic archaea, the hyperthermophile Sulfolobus solfataricus possesses a gene cluster encoding components of a putative 2-Oxoacid dehydrogenase complex. In the current paper, we have cloned and expressed the first two genes of this cluster and demonstrate that the protein products form an α2β2 hetero-tetramer possessing the catalytic activity characteristic of the first component enzyme of an acetoin dehydrogenase multienzyme complex. This represents the first report of an acetoin multienzyme complex in archaea, and contrasts with the branched-chain 2-Oxoacid dehydrogenase complex activities characterised in two other archaea, Thermoplasma acidophilum and Haloferax volcanii.
-
The 2-Oxoacid dehydrogenase multienzyme complex of Haloferax volcanii
Extremophiles, 2008Co-Authors: Dina M. Al-mailem, David W. Hough, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess four adjacent genes that, by sequence comparisons with bacteria and eukarya, appear to encode the component enzymes of a 2-Oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the archaea. In Thermoplasma acidophilum , evidence has been presented that the heterologously expressed recombinant enzyme possesses activity with the branched chain 2-Oxoacids and, to a lesser extent, with pyruvate. In the current paper, we demonstrate that in Haloferax volcanii the four genes are transcribed as an operon in vivo. However, no functional complex or individual enzyme, except for the dihydrolipoamide dehydrogenase component, could be detected in this halophile grown on a variety of carbon sources. Dihydrolipoamide dehydrogenase is present at low catalytic activities, the level of which is increased three to fourfold when Haloferax volcanii is grown on the branched-chain amino acids valine, leucine and isoleucine.
-
The 2-Oxoacid dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum - recombinant expression, assembly and characterization.
FEBS Journal, 2007Co-Authors: Caroline Heath, David W. Hough, Mareike G. Posner, Hans C. Aass, Abhishek Upadhyay, David J. Scott, Michael J. DansonAbstract:The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-Oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-Oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (Mr = 5 × 106) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-Oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-Oxoacid dehydrogenase complex.
-
discovery of the catalytic function of a putative 2 Oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, David W. Hough, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-Oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-Oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
David W. Hough - One of the best experts on this subject based on the ideXlab platform.
-
Why are the 2-Oxoacid dehydrogenase complexes so large? Generation of an active trimeric complex
Biochemical Journal, 2014Co-Authors: Nia L. Marrott, David W. Hough, Jacqueline J. T. Marshall, Dmitri I. Svergun, Susan J. Crennell, Jean M. H. Van Den Elsen, Michael J. DansonAbstract:The four-component polypeptides of the 2-Oxoacid dehydrogenase complex from the thermophilic archaeon Thermoplasma acidophilum assemble to give an active multienzyme complex possessing activity with the branched-chain 2-Oxoacids derived from leucine, isoleucine and valine, and with pyruvate. The dihydrolipoyl acyl-transferase (E2) core of the complex is composed of identical trimer-forming units that assemble into a novel 42-mer structure comprising octahedral and icosahedral geometric aspects. From our previously determined structure of this catalytic core, the inter-trimer interactions involve a tyrosine residue near the C-terminus secured in a hydrophobic pocket of an adjacent trimer like a ball-and-socket joint. In the present study, we have deleted the five C-terminal amino acids of the E2 polypeptide (IIYEI) and shown by equilibrium centrifugation that it now only assembles into a trimeric enzyme. This was confirmed by SAXS analysis, although this technique showed the presence of approximately 20% hexamers. The crystal structure of the trimeric truncated E2 core has been determined and shown to be virtually identical with the ones observed in the 42-mer, demonstrating that removal of the C-terminal anchor does not significantly affect the individual monomer or trimer structures. The truncated E2 is still able to bind both 2-Oxoacid decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components to give an active complex with catalytic activity similar to the native multienzyme complex. This is the first report of an active mini-complex for this enzyme, and raises the question of why all 2-Oxoacid dehydrogenase complexes assemble into such large structures.
-
Discovery of a putative acetoin dehydrogenase complex in the hyperthermophilic archaeon Sulfolobus solfataricus.
FEBS Letters, 2010Co-Authors: Karl A. P. Payne, David W. Hough, Michael J. DansonAbstract:Like many other aerobic archaea, the hyperthermophile Sulfolobus solfataricus possesses a gene cluster encoding components of a putative 2-Oxoacid dehydrogenase complex. In the current paper, we have cloned and expressed the first two genes of this cluster and demonstrate that the protein products form an α2β2 hetero-tetramer possessing the catalytic activity characteristic of the first component enzyme of an acetoin dehydrogenase multienzyme complex. This represents the first report of an acetoin multienzyme complex in archaea, and contrasts with the branched-chain 2-Oxoacid dehydrogenase complex activities characterised in two other archaea, Thermoplasma acidophilum and Haloferax volcanii.
-
The 2-Oxoacid dehydrogenase multienzyme complex of Haloferax volcanii
Extremophiles, 2008Co-Authors: Dina M. Al-mailem, David W. Hough, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess four adjacent genes that, by sequence comparisons with bacteria and eukarya, appear to encode the component enzymes of a 2-Oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the archaea. In Thermoplasma acidophilum , evidence has been presented that the heterologously expressed recombinant enzyme possesses activity with the branched chain 2-Oxoacids and, to a lesser extent, with pyruvate. In the current paper, we demonstrate that in Haloferax volcanii the four genes are transcribed as an operon in vivo. However, no functional complex or individual enzyme, except for the dihydrolipoamide dehydrogenase component, could be detected in this halophile grown on a variety of carbon sources. Dihydrolipoamide dehydrogenase is present at low catalytic activities, the level of which is increased three to fourfold when Haloferax volcanii is grown on the branched-chain amino acids valine, leucine and isoleucine.
-
The 2-Oxoacid dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum - recombinant expression, assembly and characterization.
FEBS Journal, 2007Co-Authors: Caroline Heath, David W. Hough, Mareike G. Posner, Hans C. Aass, Abhishek Upadhyay, David J. Scott, Michael J. DansonAbstract:The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-Oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-Oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (Mr = 5 × 106) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-Oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-Oxoacid dehydrogenase complex.
-
discovery of the catalytic function of a putative 2 Oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, David W. Hough, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-Oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-Oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
Caroline Heath - One of the best experts on this subject based on the ideXlab platform.
-
The 2-Oxoacid dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum - recombinant expression, assembly and characterization.
FEBS Journal, 2007Co-Authors: Caroline Heath, David W. Hough, Mareike G. Posner, Hans C. Aass, Abhishek Upadhyay, David J. Scott, Michael J. DansonAbstract:The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-Oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-Oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (Mr = 5 × 106) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-Oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-Oxoacid dehydrogenase complex.
-
discovery of the catalytic function of a putative 2 Oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, David W. Hough, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-Oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-Oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
-
discovery of the catalytic function of a putative 2 Oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, David W. Hough, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-Oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-Oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
-
Discovery of the catalytic function of a putative 2-Oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon Thermoplasma acidophilum.
FEBS letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, David W. Hough, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-Oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an alpha2beta2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-Oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
Peter Schadewaldt - One of the best experts on this subject based on the ideXlab platform.
-
Liver transplantation in maple syrup urine disease
European Journal of Pediatrics, 1999Co-Authors: Udo Wendel, J. M. Saudubray, A. Bodner, Peter SchadewaldtAbstract:Maple syrup urine disease (MSUD) is an autosomal recessive disorder. Impaired activity of the branched-chain 2-Oxoacid dehydrogenase complex (BCOA-DH) causes accumulation of branched-chain L-amino (BCAA) and 2-Oxoacids (BCOA) which may exert neurotoxic effects. Treatment comprises dietary management with strictly reduced quantities of protein and BCAA as well as aggressive intervention during acute neonatal and subsequent metabolic complications. MSUD is regarded as a metabolic disorder with potentially favourable outcome when the patients are kept on a carefully supervised long-term therapy. Up to now, three MSUD patients, exhibiting the classical form of the disease, have received orthotopic whole liver transplantation (OLT). Liver replacement resulted in a clear increase in whole body BCOA-DH activity to at least the level of very mild MSUD variants. These patients no longer require protein restricted diets and the risk of metabolic decompensation during catabolic events is apparently abolished. Conclusion Considering the overall expenses, risks, and outcome, however, the benefit of OLT, even in the most severe form of MSUD, may not be significantly different from that of a classical strict dietary management. Thus, OLT appears not to represent a specific option in the treatment in MSUD.
-
Liver transplantation in maple syrup urine disease
European Journal of Pediatrics, 1999Co-Authors: Udo Wendel, J. M. Saudubray, A. Bodner, Peter SchadewaldtAbstract:Maple syrup urine disease (MSUD) is an autosomal recessive disorder. Impaired activity of the branched-chain 2-Oxoacid dehydrogenase complex (BCOA-DH) causes accumulation of branched-chain L-amino (BCAA) and 2-Oxoacids (BCOA) which may exert neurotoxic effects. Treatment comprises dietary management with strictly reduced quantities of protein and BCAA as well as aggressive intervention during acute neonatal and subsequent metabolic complications. MSUD is regarded as a metabolic disorder with potentially favourable outcome when the patients are kept on a carefully supervised long-term therapy. Up to now, three MSUD patients, exhibiting the classical form of the disease, have received orthotopic whole liver transplantation (OLT). Liver replacement resulted in a clear increase in whole body BCOA-DH activity to at least the level of very mild MSUD variants. These patients no longer require protein restricted diets and the risk of metabolic decompensation during catabolic events is apparently abolished.
Victoria I. Bunik - One of the best experts on this subject based on the ideXlab platform.
-
the 2 Oxoacid dehydrogenase complexes in mitochondria can produce superoxide hydrogen peroxide at much higher rates than complex i
Journal of Biological Chemistry, 2014Co-Authors: Casey L. Quinlan, Victoria I. Bunik, Renata L.s. Goncalves, Nagendra Yadava, Martin Heymogensen, Martin D. BrandAbstract:Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD+ or NADH at about the same operating redox potential as the NADH/NAD+ pool and comprise the NADH/NAD+ isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-Oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)+ ratios, although the 2-Oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-Oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.
-
The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates than Complex I
Journal of Biological Chemistry, 2014Co-Authors: Casey L. Quinlan, Victoria I. Bunik, Renata L.s. Goncalves, Martin Hey-mogensen, Nagendra Yadava, Martin D. BrandAbstract:Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD+ or NADH at about the same operating redox potential as the NADH/NAD+ pool and comprise the NADH/NAD+ isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-Oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)+ ratios, although the 2-Oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-Oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.
-
Interaction of thioredoxins with target proteins: role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2-Oxoacid dehydrogenase complexes.
Protein Science, 2008Co-Authors: Victoria I. Bunik, Yves Meyer, Jean-pierre Jacquot, Günter Raddatz, Stéphane D. Lemaire, Hans BisswangerAbstract:The thioredoxin action upon the 2-Oxoacid dehydrogenase complexes is investigated by using different thioredoxins, both wild-type and mutated. The attacking cysteine residue of thioredoxin is established to be essential for the thioredoxin-dependent activation of the complexes. Mutation of the buried cysteine residue to serine is not crucial for the activation, but prevents inhibition of the complexes, exhibited by the Clamydomonas reinhardtii thioredoxin m disulfide. Site-directed mutagenesis of D26, W31, F/W12, and Y/A70 (the Escherichia coli thioredoxin numbering is employed for all the thioredoxins studied) indicates that both the active site and remote residues of thioredoxin are involved in its interplay with the 2-Oxoacid dehydrogenase complexes. Sequences of 11 thioredoxin species tested biochemically are aligned. The thioredoxin residues at the contact between the alpha3/3(10) and alpha1 helices, the length of the alpha1 helix and the charges in the alpha2-beta3 and beta4-beta5 linkers are found to correlate with the protein influence on the 2-Oxoacid dehydrogenase complexes (the secondary structural elements of thioredoxin are defined according to Eklund H et al., 1991, Proteins 11:13-28). The distribution of the charges on the surface of the thioredoxin molecules is analyzed. The analysis reveals the species specific polarization of the thioredoxin active site surroundings, which corresponds to the efficiency of the thioredoxin interplay with the 2-Oxoacid dehydrogenase systems. The most effective mitochondrial thioredoxin is characterized by the strongest polarization of this area and the highest value of the electrostatic dipole vector of the molecule. Not only the magnitude, but also the orientation of the dipole vector show correlation with the thioredoxin action. The dipole direction is found to be significantly influenced by the charges of the residues 13/14, 51, and 83/85, which distinguish the activating and inhibiting thioredoxin disulfides.
-
Activation of mitochondrial 2-Oxoacid dehydrogenases by thioredoxin.
Biological Chemistry, 1997Co-Authors: Victoria I. Bunik, Hartmut Follmann, Hans BisswangerAbstract:The regulation of mitochondrial dehydrogenases of 2-Oxoacids by thioredoxin is established. It is found that at low NAD+ and saturating concentrations of 2-Oxoacids and CoA, inactivation of 2-Oxoacid dehydrogenase complexes takes place, preventing NAD+ reduction under such conditions. However, addition of oxidized E. coli thioredoxin to the reaction medium without dithiothreitol allows effective NAD+ reduction at this substrate ratio. Product accumulation curves show that thioredoxin activates the complexes by protecting them from the inactivation observed in the conditions when the complex-bound dihydrolipoate is accumulated. Disappearance of the activatory effect of thioredoxin after its treatment with SH-specific reagents indicates the involvement of the redox-active cysteine couple of thioredoxin in its activation of 2-Oxoacid dehydrogenase complexes. The redox-inactive thioredoxin not only shows no activation, but in fact exerts an inhibitory effect. The inhibition manifests the complex formation between SH-modified thioredoxin and dehydrogenase systems, involving amino acid residues of thioredoxin other than cysteine. High efficiency of thioredoxin from E. coli as compared to chloroplast thioredoxin f and glutathione disulfide is revealed. This indicates the importance of specific protein structure also for the influence of the redox-active thioredoxin upon the 2-Oxoacid dehydrogenase complexes. The results obtained suggest that these key enzyme systems of mitochondrial metabolism represent previously unidentified targets for the action of mitochondrial thioredoxin, which is known to resemble the E. coli counterpart studies in this work.
-
Using lipoate enantiomers and thioredoxin to study the mechanism of the 2-Oxoacid-dependent dihydrolipoate production by the 2-Oxoacid dehydrogenase complexes
FEBS Letters, 1995Co-Authors: Victoria I. Bunik, Hans Bisswanger, A. Shoubnikova, S. Loeffelhardt, H.o. Borbe, Hartmut FollmannAbstract:The thioredoxin-catalyzed insulin reduction by dihydrolipoate was applied to study the 2-Oxoacid:lipoate oxidoreductase activity of 2-Oxoacid dehydrogenase complexes. The enzymatic and non-enzymatic mechanisms of the transfer of reducing equivalents from the complexes to free lipoic acid (α-lipoic acid, 6,8-thiooctic acid) were distinguished using the high stereoselectivity of the complex enzymes to the R-enantiomer of lipoate. Unlike these enzymes, thioredoxin from E. coli exibited no stereoselectivity upon reduction with chemically obtained dihydrolipoate. However, coupled to the dihydrolipoate production by dehydrogenase complexes, the process was essentially sensitive both to the enantiomer used and the dihydrolipoyl dehydrogenase activity of the complexes. These results indicated the involvement of the third complex component, dihydrolipoyl dehydrogenase, in the 2-Oxoacid-dependent dihydrolipoate formation. The implication of the investigated reaction for a connection between thioredoxin and the 2-Oxoacid dehydrogenase complexes in the mitochondrial metabolism are discussed.
