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Michael J. Danson – One of the best experts on this subject based on the ideXlab platform.

  • Comparative Genomic Analysis Reveals 2Oxoacid Dehydrogenase Complex Lipoylation Correlation with
    , 2016
    Co-Authors: Aerobiosis In Archaea, Michael J. Danson, Mareike G. Posner, Abhishek Upadhyay, Kirill Borziak, Stefan Bagby, Steve Dorus

    Abstract:

    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 2Oxoacid 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

  • Why are the 2Oxoacid Dehydrogenase complexes so large? Generation of an active trimeric complex
    Biochemical Journal, 2014
    Co-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. Danson

    Abstract:

    The four-component polypeptides of the 2Oxoacid Dehydrogenase complex from the thermophilic archaeon Thermoplasma acidophilum assemble to give an active multienzyme complex possessing activity with the branched-chain 2Oxoacids 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 2Oxoacid 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 2Oxoacid Dehydrogenase complexes assemble into such large structures.

  • Comparative genomic analysis reveals 2Oxoacid Dehydrogenase complex lipoylation correlation with aerobiosis in archaea.
    PLoS ONE, 2014
    Co-Authors: Kirill Borziak, Michael J. Danson, Mareike G. Posner, Abhishek Upadhyay, Stefan Bagby, Steve Dorus

    Abstract:

    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 2Oxoacid 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.

David W. Hough – One of the best experts on this subject based on the ideXlab platform.

  • Why are the 2Oxoacid Dehydrogenase complexes so large? Generation of an active trimeric complex
    Biochemical Journal, 2014
    Co-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. Danson

    Abstract:

    The four-component polypeptides of the 2Oxoacid Dehydrogenase complex from the thermophilic archaeon Thermoplasma acidophilum assemble to give an active multienzyme complex possessing activity with the branched-chain 2Oxoacids 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 2Oxoacid 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 2Oxoacid Dehydrogenase complexes assemble into such large structures.

  • the catalytic core of an archaeal 2 Oxoacid Dehydrogenase multienzyme complex is a 42 mer protein assembly
    FEBS Journal, 2012
    Co-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 Elsen

    Abstract:

    The dihydrolipoyl acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2Oxoacid 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)

  • The catalytic core of an archaeal 2Oxoacid Dehydrogenase multienzyme complex is a 42‐mer protein assembly
    FEBS Journal, 2012
    Co-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 Elsen

    Abstract:

    The dihydrolipoyl acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2Oxoacid 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)

Caroline Heath – One of the best experts on this subject based on the ideXlab platform.

  • The 2Oxoacid Dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum – recombinant expression, assembly and characterization.
    FEBS Journal, 2007
    Co-Authors: Caroline Heath, David W. Hough, Mareike G. Posner, Hans C. Aass, Abhishek Upadhyay, David J. Scott, Michael J. Danson

    Abstract:

    The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2Oxoacid Dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2Oxoacid 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 2Oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2Oxoacid Dehydrogenase complex.

  • discovery of the catalytic function of a putative 2 Oxoacid Dehydrogenase multienzyme complex in the thermophilic archaeon thermoplasma acidophilum
    FEBS Letters, 2004
    Co-Authors: Caroline Heath, Alex C Jeffries, David W. Hough, Michael J. Danson

    Abstract:

    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 2Oxoacid 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 2Oxoacid 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, 2004
    Co-Authors: Caroline Heath, Alex C Jeffries, David W. Hough, Michael J. Danson

    Abstract:

    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 2Oxoacid 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 2Oxoacid Dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.