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Michael J. Danson - One of the best experts on this subject based on the ideXlab platform.
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Comparative Genomic Analysis Reveals 2-Oxoacid Dehydrogenase Complex Lipoylation Correlation with
2016Co-Authors: Aerobiosis In Archaea, Michael J. Danson, Mareike G. Posner, Abhishek Upadhyay, Kirill Borziak, Stefan Bagby, Steve DorusAbstract:Metagenomic analyses have advanced our understanding of ecological microbial diversity, but to what extent can metagenomic data be used to predict the metabolic capacity of difficult-to-study organisms and their abiotic environmental interactions? We tackle this question, using a comparative genomic approach, by considering the molecular basis of aerobiosis within archaea. Lipoylation, the covalent attachment of lipoic acid to 2-Oxoacid dehydrogenase multienzyme complexes (OADHCs), is essential for metabolism in aerobic bacteria and eukarya. Lipoylation is catalysed either by lipoate protein ligase (LplA), which in archaea is typically encoded by two genes (LplA-N and LplA-C), or by a lipoyl(octanoyl) transferase (LipB or LipM) plus a lipoic acid synthetase (LipA). Does the genomic presence of lipoylation and OADHC genes across archaea from diverse habitats correlate with aerobiosis? First, analyses of 11,826 biotin protein ligase (BPL)-LplA-LipB transferase family members and 147 archaeal genomes identified 85 species with lipoylation capabilities and provided support for multiple ancestral acquisitions of lipoylation pathways during archaeal evolution. Second, with the exception of the Sulfolobales order, the majority of species possessing lipoylation systems exclusively retain LplA, or either LipB or LipM, consistent with archaeal genome streamlining. Third, obligate anaerobic archaea display widespread loss of lipoylation and OADHC genes. Conversely, a high level of correspondence is observed between aerobiosis and the presence of LplA/LipB/ LipM, LipA and OADHC E2, consistent with the role of lipoylation in aerobic metabolism. This correspondence betwee
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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.
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Comparative genomic analysis reveals 2-Oxoacid dehydrogenase complex lipoylation correlation with aerobiosis in archaea.
PLoS ONE, 2014Co-Authors: Kirill Borziak, Michael J. Danson, Mareike G. Posner, Abhishek Upadhyay, Stefan Bagby, Steve DorusAbstract:Metagenomic analyses have advanced our understanding of ecological microbial diversity, but to what extent can metagenomic data be used to predict the metabolic capacity of difficult-to-study organisms and their abiotic environmental interactions? We tackle this question, using a comparative genomic approach, by considering the molecular basis of aerobiosis within archaea. Lipoylation, the covalent attachment of lipoic acid to 2-Oxoacid dehydrogenase multienzyme complexes (OADHCs), is essential for metabolism in aerobic bacteria and eukarya. Lipoylation is catalysed either by lipoate protein ligase (LplA), which in archaea is typically encoded by two genes (LplA-N and LplA-C), or by a lipoyl(octanoyl) transferase (LipB or LipM) plus a lipoic acid synthetase (LipA). Does the genomic presence of lipoylation and OADHC genes across archaea from diverse habitats correlate with aerobiosis? First, analyses of 11,826 biotin protein ligase (BPL)-LplA-LipB transferase family members and 147 archaeal genomes identified 85 species with lipoylation capabilities and provided support for multiple ancestral acquisitions of lipoylation pathways during archaeal evolution. Second, with the exception of the Sulfolobales order, the majority of species possessing lipoylation systems exclusively retain LplA, or either LipB or LipM, consistent with archaeal genome streamlining. Third, obligate anaerobic archaea display widespread loss of lipoylation and OADHC genes. Conversely, a high level of correspondence is observed between aerobiosis and the presence of LplA/LipB/LipM, LipA and OADHC E2, consistent with the role of lipoylation in aerobic metabolism. This correspondence between OADHC lipoylation capacity and aerobiosis indicates that genomic pathway profiling in archaea is informative and that well characterized pathways may be predictive in relation to abiotic conditions in difficult-to-study extremophiles. Given the highly variable retention of gene repertoires across the archaea, the extension of comparative genomic pathway profiling to broader metabolic and homeostasis networks should be useful in revealing characteristics from metagenomic datasets related to adaptations to diverse environments.
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the catalytic core of an archaeal 2 Oxoacid dehydrogenase multienzyme complex is a 42 mer protein assembly
FEBS Journal, 2012Co-Authors: Nia L. Marrott, Michael J. Danson, David W. Hough, Jacqueline J. T. Marshall, Dmitri I. Svergun, Susan J. Crennell, Jean M. H. Van Den ElsenAbstract:The dihydrolipoyl acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2-Oxoacid dehydrogenase multienzyme complexes of the central metabolic pathways. Although this family of multienzyme complexes shares a common architecture, their E2 cores form homo-trimers that, depending on the source, further associate into either octahedral (24-mer) or icosahedral (60-mer) assemblies, as predicted by the principles of quasi-equivalence. In the crystal structure of the E2 core from Thermoplasma acidophilum, a thermophilic archaeon, the homo-trimers assemble into a unique 42-mer oblate spheroid. Analytical equilibrium centrifugation and small-angle X-ray scattering analyses confirm that this catalytically active 1.08 MDa assembly exists as a single species in solution, forming a hollow spheroid with a maximum diameter of 220 A. In this paper we show that a monodisperse macromolecular assembly, built from identical subunits in non-identical environments, forms an irregular protein shell via non-equivalent interactions. This unusually irregular protein shell, combining cubic and dodecahedral geometrical elements, expands on the concept of quasi-equivalence as a basis for understanding macromolecular assemblies by showing that cubic point group symmetry is not a physical requirement in multienzyme assembly. These results extend our basic knowledge of protein assembly and greatly expand the number of possibilities to manipulate self-assembling biological complexes to be utilized in innovative nanotechnology applications. Database The final coordinates of the E2 structure have been deposited in the Protein Data Bank (PDB accession code 3RQC) Structured digital abstract • E2 and E2 bind by x-ray crystallography (View interaction) • E2 and E2 bind by x ray scattering (View interaction)
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The catalytic core of an archaeal 2‐Oxoacid dehydrogenase multienzyme complex is a 42‐mer protein assembly
FEBS Journal, 2012Co-Authors: Nia L. Marrott, Michael J. Danson, David W. Hough, Jacqueline J. T. Marshall, Dmitri I. Svergun, Susan J. Crennell, Jean M. H. Van Den ElsenAbstract:The dihydrolipoyl acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2-Oxoacid dehydrogenase multienzyme complexes of the central metabolic pathways. Although this family of multienzyme complexes shares a common architecture, their E2 cores form homo-trimers that, depending on the source, further associate into either octahedral (24-mer) or icosahedral (60-mer) assemblies, as predicted by the principles of quasi-equivalence. In the crystal structure of the E2 core from Thermoplasma acidophilum, a thermophilic archaeon, the homo-trimers assemble into a unique 42-mer oblate spheroid. Analytical equilibrium centrifugation and small-angle X-ray scattering analyses confirm that this catalytically active 1.08 MDa assembly exists as a single species in solution, forming a hollow spheroid with a maximum diameter of 220 A. In this paper we show that a monodisperse macromolecular assembly, built from identical subunits in non-identical environments, forms an irregular protein shell via non-equivalent interactions. This unusually irregular protein shell, combining cubic and dodecahedral geometrical elements, expands on the concept of quasi-equivalence as a basis for understanding macromolecular assemblies by showing that cubic point group symmetry is not a physical requirement in multienzyme assembly. These results extend our basic knowledge of protein assembly and greatly expand the number of possibilities to manipulate self-assembling biological complexes to be utilized in innovative nanotechnology applications. Database The final coordinates of the E2 structure have been deposited in the Protein Data Bank (PDB accession code 3RQC) Structured digital abstract • E2 and E2 bind by x-ray crystallography (View interaction) • E2 and E2 bind by x ray scattering (View interaction)
Victoria I. Bunik - One of the best experts on this subject based on the ideXlab platform.
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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.
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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.
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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.
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Structural determinants for the efficient and specific interaction of thioredoxin with 2-Oxoacid dehydrogenase complexes
Applied Biochemistry and Biotechnology, 2000Co-Authors: Günter Raddatz, Volker Kruft, Victoria I. BunikAbstract:Specificity and efficiency of thiored oxin action upon the 2-Oxoacid dehydrogenase complexes are studied by using a number of thiored oxins and complexes. Bacterial and mammalian pyruvate and 2-oxoglutarate dehydrogenase systems display similar row of preference to thioredoxins that may result from thioredoxin binding to the homologous or common dihydrolipoamide dehydrogenase components of the complexes. The most sensitive tothioredoxin is the complex whose component exhibits the highest sequence similarity to eukaryotic thioredoxin reductase. Hence, thioredoxin binding to the complexes may be related to that in the thioredoxin reductase, a dihydrolipoamide dehydrogenase homolog. The highest potency of mitochondrial thioredoxin to affect the mitochondiral complexes is revealed. A 96–100% conservation of the mitochondrial thioredoxin structure is shown within the four known sequences and the N-terminus of the pigheart protein determined. Eleven thioredoxins tested biochemically are analyzed by multiple sequence alignment and homology modeling. Their effects correlate with the residues at the contact between the α 3/3_10 and α 1 helices, the length of the α 1 helix and charges in the α2–β3 and β4–β5 linkers. Polarization of the thioredoxin molecule and its active site surroundings are characterized. Thioredoxins with a highly polarized surface around the essential disulfide bridge (mitochondiral, pea f , and Arabidopsis thaliana h3 ) show low cross-reactivity as compared to the species with a decreased polarization of this area (e.g., from Escherichia coli ). The strongest polarization of the whole molecule results in the highest magnitude of the electrostatic dipole vector of mitochondrial thioredoxin. Thiored oxins with the dipole orientation similar to that of the latter have the affinities for the 2-Oxoacid dehydrogenase complexes, proportional to the dipole magnitudes. Thioredoxin with an opposite dipole orientation shows no effect. Activating and inhibitory thioredoxin disulfides are distinguished by the charges of the residues 13/14 (α1 helix(, 51 (α2–β3 linker), and 83/85 (β4–β5 linker), changing the dipole direction. The results show that the thioredoxin-target interplay may be controlled by the long-range interactions between the electrostatic dipole vectors of the proteins and the degree of their interface polarization.
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Increased catalytic performance of the 2-Oxoacid dehydrogenase complexes in the presence of thioredoxin, a thiol–disulfide oxidoreductase
Journal of Molecular Catalysis B-enzymatic, 2000Co-Authors: Victoria I. BunikAbstract:Abstract Bacterial and mammalian pyruvate and 2-oxoglutarate dehydrogenase complexes undergo an irreversible inactivation upon accumulation of the dihydrolipoate intermediate. The first component of the complexes, 2-Oxoacid dehydrogenase, is affected. Addition of thioredoxin protects from this inactivation, increasing catalytic rates and limiting degrees of the substrate transformation to products, acyl-CoA and NADH. Although the redox active cysteines of thioredoxin are essential for its interplay with the complexes, the effects are observed with both dithiol and disulfide forms of the protein. This indicates that thioredoxin affects an SH/S–S component of the system, which is present in the two redox states. The complex-bound lipoate is concluded to be the thioredoxin target, since (i) both dithiol and disulfide forms of the residue are available during the catalytic cycle and (ii) the thioredoxin reaction with the essential SH/S–S group of the terminal component of the complex, dihydrolipoyl dehydrogenase, is excluded. Thus, the thioredoxin disulfide interacts with the dihydrolipoate intermediate, while the thioredoxin dithiol reacts with the lipoate disulfide. Kinetic consequences of such interplay are consistent with the observed thioredoxin effects. Owing to the essential reactivity of the SH/S–S couple in thioredoxin, the thiol–disulfide exchange between thioredoxin and the lipoate residue is easy reversible, providing both protection (by the mixed disulfide formation) and catalysis (by the appropriate lipoate release). In contrast, non-protein SH/S–S compounds prevent the inactivatory action of dihydrolipoate intermediate only at a high excess over the complex-bound lipoate. This interferes with the catalysis-required release of the residue from its mixed disulfide. Therefore, only thioredoxin is capable to `buffer' the steady-state concentration of the reactive dithiol. Such action represents a new thioredoxin function, which may be exploited to protect other enzymes with exposed redox-active thiol intermediates.
David W. Hough - One of the best experts on this subject based on the ideXlab platform.
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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.
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the catalytic core of an archaeal 2 Oxoacid dehydrogenase multienzyme complex is a 42 mer protein assembly
FEBS Journal, 2012Co-Authors: Nia L. Marrott, Michael J. Danson, David W. Hough, Jacqueline J. T. Marshall, Dmitri I. Svergun, Susan J. Crennell, Jean M. H. Van Den ElsenAbstract:The dihydrolipoyl acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2-Oxoacid dehydrogenase multienzyme complexes of the central metabolic pathways. Although this family of multienzyme complexes shares a common architecture, their E2 cores form homo-trimers that, depending on the source, further associate into either octahedral (24-mer) or icosahedral (60-mer) assemblies, as predicted by the principles of quasi-equivalence. In the crystal structure of the E2 core from Thermoplasma acidophilum, a thermophilic archaeon, the homo-trimers assemble into a unique 42-mer oblate spheroid. Analytical equilibrium centrifugation and small-angle X-ray scattering analyses confirm that this catalytically active 1.08 MDa assembly exists as a single species in solution, forming a hollow spheroid with a maximum diameter of 220 A. In this paper we show that a monodisperse macromolecular assembly, built from identical subunits in non-identical environments, forms an irregular protein shell via non-equivalent interactions. This unusually irregular protein shell, combining cubic and dodecahedral geometrical elements, expands on the concept of quasi-equivalence as a basis for understanding macromolecular assemblies by showing that cubic point group symmetry is not a physical requirement in multienzyme assembly. These results extend our basic knowledge of protein assembly and greatly expand the number of possibilities to manipulate self-assembling biological complexes to be utilized in innovative nanotechnology applications. Database The final coordinates of the E2 structure have been deposited in the Protein Data Bank (PDB accession code 3RQC) Structured digital abstract • E2 and E2 bind by x-ray crystallography (View interaction) • E2 and E2 bind by x ray scattering (View interaction)
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The catalytic core of an archaeal 2‐Oxoacid dehydrogenase multienzyme complex is a 42‐mer protein assembly
FEBS Journal, 2012Co-Authors: Nia L. Marrott, Michael J. Danson, David W. Hough, Jacqueline J. T. Marshall, Dmitri I. Svergun, Susan J. Crennell, Jean M. H. Van Den ElsenAbstract:The dihydrolipoyl acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2-Oxoacid dehydrogenase multienzyme complexes of the central metabolic pathways. Although this family of multienzyme complexes shares a common architecture, their E2 cores form homo-trimers that, depending on the source, further associate into either octahedral (24-mer) or icosahedral (60-mer) assemblies, as predicted by the principles of quasi-equivalence. In the crystal structure of the E2 core from Thermoplasma acidophilum, a thermophilic archaeon, the homo-trimers assemble into a unique 42-mer oblate spheroid. Analytical equilibrium centrifugation and small-angle X-ray scattering analyses confirm that this catalytically active 1.08 MDa assembly exists as a single species in solution, forming a hollow spheroid with a maximum diameter of 220 A. In this paper we show that a monodisperse macromolecular assembly, built from identical subunits in non-identical environments, forms an irregular protein shell via non-equivalent interactions. This unusually irregular protein shell, combining cubic and dodecahedral geometrical elements, expands on the concept of quasi-equivalence as a basis for understanding macromolecular assemblies by showing that cubic point group symmetry is not a physical requirement in multienzyme assembly. These results extend our basic knowledge of protein assembly and greatly expand the number of possibilities to manipulate self-assembling biological complexes to be utilized in innovative nanotechnology applications. Database The final coordinates of the E2 structure have been deposited in the Protein Data Bank (PDB accession code 3RQC) Structured digital abstract • E2 and E2 bind by x-ray crystallography (View interaction) • E2 and E2 bind by x ray scattering (View interaction)
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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.
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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.
Caroline Heath - One of the best experts on this subject based on the ideXlab platform.
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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.
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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.
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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.
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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.
Takayoshi Wakagi - One of the best experts on this subject based on the ideXlab platform.
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crystal structures of archaeal 2 Oxoacid ferredoxin oxidoreductases from sulfolobus tokodaii
Scientific Reports, 2016Co-Authors: Zhen Yan, Akane Maruyama, Takatoshi Arakawa, Shinya Fushinobu, Takayoshi WakagiAbstract:As the first three-dimensional structure of the two-subunit type 2-Oxoacid:ferredoxin oxidoreductases (OFOR) from archaea, we solved the crystal structures of STK_23000/STK_22980 (StOFOR1) and STK_24350/STK_24330 (StOFOR2) from Sulfolobus tokodaii. They showed similar overall structures, consisting of two a- and b-subunit heterodimers containing thiamin pyrophosphate (TPP) cofactor and [4Fe-4S] cluster, but lack an intramolecular ferredoxin domain. Unlike other OFORs, StOFORs can utilize both pyruvate and 2-oxoglutarate, playing a key role in the central metabolism. In the structure of StOFOR2 in unreacted pyruvate complex form, carboxylate group of pyruvate is recognized by Arg344 and Thr257 from the a-subunit, which are conserved in pyruvate:ferredoxin oxidoreductase from Desulfovbrio africanus (DaPFOR). In the structure of StOFOR1 co-crystallized with 2-oxobutyrate, electron density corresponding to a 1-hydroxypropyl group (post-decarboxylation state) was observed at the thiazole ring of TPP. The binding pockets of the StOFORs surrounding the methyl or propyl group of the ligands are wider than that of DaPFOR. Mutational analyses indicated that several residues were responsible for the broad 2-Oxoacid specificity of StOFORs. We also constructed a possible complex structural model by placing a Zn(2+)-containing dicluster ferredoxin of S. tokodaii into the large pocket of StOFOR2, providing insight into the electron transfer between the two redox proteins.
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Substrate recognition by 2-Oxoacid:ferredoxin oxidoreductase from Sulfolobus sp. strain 7.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2002Co-Authors: Eriko Fukuda, Takayoshi WakagiAbstract:Abstract 2-Oxoacid:ferredoxin oxidoreductase (OFOR) catalyzes the coenzyme A-dependent oxidative decarboxylation of 2-Oxoacids, at an analogous metabolic position to 2-Oxoacid dehydrogenase multienzyme complex. The enzyme from Sulfolobus sp. strain 7, a thermoacidophilic crenarchaeon, is a heterodimer comprising two subunits, a (632 amino acids) and b (305 amino acids). In contrast to other OFORs, the Sulfolobus enzyme shows a broad specificity for 2-Oxoacids such as pyruvate and 2-oxoglutarate. Based on careful multiple alignment of this enzyme family and on the reported three-dimensional structure of the homodimeric pyruvate:ferredoxin oxidoreductase (POR) from Desulfovibrio africanus , we selected five amino acids, T256, R344 and T353 of subunit-a, and K49 and L123 of subunit-b, as candidate 2-Oxoacid recognizing residues. To identify the residues determining the 2-Oxoacid specificity of the enzyme family, we performed point mutations of these five amino acids, and characterized the resulting mutants. Analyses of the mutants revealed that R344 of subunit-a of the enzyme was essential for the activity, and that K49R and L123N of subunit-b drastically affected the enzyme specificity for pyruvate and 2-oxoglutarate, respectively. Replacement of the five residues resulted in significant changes in both K m and V max , indicating that these amino acids are clearly involved in substrate recognition and catalysis.
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role of a highly conserved ypitp motif in 2 Oxoacid ferredoxin oxidoreductase heterologous expression of the gene from sulfolobus sp strain 7 and characterization of the recombinant and variant enzymes
FEBS Journal, 2001Co-Authors: Eriko Fukuda, Hiroyasu Kino, Hiroshi Matsuzawa, Takayoshi WakagiAbstract:2-Oxoacid:ferredoxin oxidoreductase from Sulfolobus sp. strain 7, an aerobic and thermoacidophilic crenoarchaeon, catalyses the coenzyme A-dependent oxidative decarboxylation of pyruvate and 2-oxoglutarate, a cognate Zn-7Fe-ferredoxin serving as an electron acceptor. It comprises two subunits, a (632 amino acids) and b (305 amino acids). To further elucidate its structure and function, we constructed a gene expression system. The wild-type recombinant enzyme was indistinguishable from the natural one in every criterion investigated. A series of variants was constructed to elucidate the role of the YPITP-motif (residues 253-257) in subunit a, which is conserved universally in the 2-Oxoacid:ferredoxin oxidoreductase (OFOR) family. Single amino-acid replacements at Y253 and P257 by other amino acids caused a drastic loss of enzyme activity. T256, the hydroxyl group of which has been proposed to be essential for binding of the 2-oxo group of the substrate in the Desulfovibrio africanus enzyme, was unexpectedly replaceable with Ala, the kcat and Km for 2-oxoglutarate being approximately 33% and approximately 51%, respectively, as compared with that of the wild-type enzyme. Replacement at other positions resulted in a significant decrease in the kcat of the reaction while the Km for 2-Oxoacid was only slightly affected. Thus, the YPITP-motif is essential for the turnover of the reaction rather than the affinity toward 2-Oxoacid.
