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Richard N. Perham - One of the best experts on this subject based on the ideXlab platform.
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the role of loop and β turn residues as structural and functional determinants for the lipoyl domain from the escherichia coli 2 oxoglutarate dehydrogenase complex
Biochemical Journal, 2008Co-Authors: Darran Dafydd Jones, Richard N. PerhamAbstract:The lipoyl domain of the dihydrolipoyl succinyltransferase (E2o) component of the 2OGDH (2-oxoglutarate dehydrogenase) multienzyme complex houses the lipoic acid cofactor through covalent attachment to a specific lysine side chain residing at the tip of a b-turn. Residues within the lipoyl-lysine b-turn and a nearby prominent loop have been implicated as determinants of lipoyl domain structure and function. Protein engineering of the Escherichia coli E2o lipoyl domain (E2olip) revealed that removal of residues from the loop caused a major structural change in the protein, which rendered the domain incapable of reductive succinylation by 2-oxoglutarate decarboxylase (E1o) and reduced the Lipoylation efficiency. Insertion of a new loop corresponding to that of the E. coli pyruvate dehydrogenase lipoyl domain (E2plip) restored Lipoylation efficiency and the capacity to undergo reductive succinylation returned, albeit at a lower rate. Exchange of the E2olip loop sequence significantly improved the ability of the domain to be reductively acetylated by pyruvate decarboxylase (E1p), retaining approx. 10-fold more acetyl groups after 25 min than wild-type E2olip. Exchange of the b-turn residue on the N-terminal side of the E2o lipoyl-lysine DKA/V motif to the equivalent residue in E2plip (T42G), both singly and in conjunction with the loop exchange, reduced the ability of the domain to be reductively succinylated, but led to an increased capacity to be reductively acetylated by the non-cognate E1p. The T42G mutation also slightly enhanced the Lipoylation rate of the domain. The surface loop is important to the structural integrity of the protein and together with Thr42 plays an important role in specifying the interaction of the lipoyl domain with its partner E1o in the E. coli 2OGDH complex.
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42 structure and posttranslational modification of lipoyl domain of 2 oxo acid dehydrogenase multienzyme complexes
Methods in Enzymology, 1995Co-Authors: Richard N. PerhamAbstract:Publisher Summary This chapter discusses the structure and posttranslational modification of lipoyl domain of 2-oxo-acid dehydrogenase multienzyme complexes. 2-oxo-acid dehydrogenase complexes catalyze the oxidative decarboxylation of a specific 2-oxo-acid and the subsequent acylation of coenzyme A, with the concomitant formation of NADH. A typical pyruvate dehydrogenase complex consists of multiple copies of three enzymes that act sequentially: pyruvate decarboxylase, dihydrolipoyl acetyltransferase, and dihydrolipoyl dehydrogenase. The E2 component is a multidomain polypeptide chain that aggregates with octahedral (24-mer) or icosahedral (60-mer) symmetry, depending on the source. The symmetry is dictated by the large C-terminal domain that also houses the acyltransferase active site. The N-terminal half of the polypeptide chain comprises one or more lipoyl domains and a peripheral subunit (E1/E3)-binding domain joined together by long and highly flexible linker regions of polypeptide chain. 2-oxo-acid dehydrogenase complexes specific for the oxidative decarboxylation of other 2-oxo acids—such as 2-oxoglutarate and branched-chain 2-oxo acids derived from the transamination of valine, leucine, and isoleucine—follow similar structural patterns. This chapter describes the purification of the lipoyl domain of the B. stearothermophilus pyruvate dehydrogenase complex and summarizes its solution structure. The chapter also describes the mechanism of posttranslational modification of the lipoyl domain, in particular, of the selection of the target lysine residue for Lipoylation.
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Lipoylation of the e2 components of the 2 oxo acid dehydrogenase multienzyme complexes of escherichia coli
Biochemical Journal, 1991Co-Authors: Leonard C Packman, B Green, Richard N. PerhamAbstract:The number of functional lipoyl groups in the dihydrolipoyl acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex from Escherichia coli has been re-assessed by means of a combination of protein-chemical and mass-spectrometric techniques. (1) After the complex had been treated with N-ethyl[2,3-14C]maleimide in the presence of pyruvate, the lipoyl domains were excised from the complex, treated with NaBH4 and re-exposed to N-ethyl[2,3-14C]maleimide. All the chemically reactive lipoyl groups in the native complex were found to be catalytically active. (2) Proteolytic digests of the separated lipoyl domains were examined for the presence of the Lipoylation-site peptide, GDKASME, with and without the lipoyl group in N6-linkage to the lysine residue. Only the lipoylated form of the peptide was detected, suggesting that all three lipoyl domains are fully substituted at this site. (3) The behaviour of each lipoyl domain was examined on ion-exchange chromatography in response to alkylation with 4-vinylpyridine after either chemical reduction of the lipoyl group with dithiothreitol or reductive acetylation by the pyruvate dehydrogenase complex in the presence of pyruvate. All three domains exhibited a quantitative shift in retention time, confirming that each domain was fully substituted by an enzymically reactive lipoyl group. (4) When subjected to electrospray mass spectrometry, each domain gave a mass consistent with a fully lipoylated domain, and no aberrant substitution of the target lysine residue was detected. The same result was obtained for the lipoyl domain from the E. coli 2-oxoglutarate dehydrogenase complex. (5) Previous widespread attempts to assess the number of functional lipoyl groups in the pyruvate dehydrogenase multienzyme complex, which have led to the view that a maximum of two lipoyl groups per E2 chain may be involved in the catalytic mechanism, are in error.
Sylke Muller - One of the best experts on this subject based on the ideXlab platform.
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knockout studies reveal an important role of plasmodium lipoic acid protein ligase a1 for asexual blood stage parasite survival
PLOS ONE, 2009Co-Authors: Svenja Gunther, Kai Matuschewski, Sylke MullerAbstract:Lipoic acid (LA) is a dithiol-containing cofactor that is essential for the function of α-keto acid dehydrogenase complexes. LA acts as a reversible acyl group acceptor and ‘swinging arm’ during acyl-coenzyme A formation. The cofactor is post-translationally attached to the acyl-transferase subunits of the multienzyme complexes through the action of octanoyl (lipoyl): N-octanoyl (lipoyl) transferase (LipB) or lipoic acid protein ligases (LplA). Remarkably, apicomplexan parasites possess LA biosynthesis as well as scavenging pathways and the two pathways are distributed between mitochondrion and a vestigial organelle, the apicoplast. The apicoplast-specific LipB is dispensable for parasite growth due to functional redundancy of the parasite's lipoic acid/octanoic acid ligases/transferases. In this study, we show that LplA1 plays a pivotal role during the development of the erythrocytic stages of the malaria parasite. Gene disruptions in the human malaria parasite P. falciparum consistently were unsuccessful while in the rodent malaria model parasite P. berghei the LplA1 gene locus was targeted by knock-in and knockout constructs. However, the LplA1(−) mutant could not be cloned suggesting a critical role of LplA1 for asexual parasite growth in vitro and in vivo. These experimental genetics data suggest that Lipoylation during expansion in red blood cells largely occurs through salvage from the host erythrocytes and subsequent ligation of LA to the target proteins of the malaria parasite.
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apicoplast lipoic acid protein ligase b is not essential for plasmodium falciparum
PLOS Pathogens, 2007Co-Authors: Svenja Gunther, Lynsey J M Wallace, Evamaria Patzewitz, Paul J Mcmillan, Janet Storm, Carsten Wrenger, Ryan Bissett, Terry K Smith, Sylke MullerAbstract:Lipoic acid (LA) is an essential cofactor of α-keto acid dehydrogenase complexes (KADHs) and the glycine cleavage system. In Plasmodium, LA is attached to the KADHs by organelle-specific Lipoylation pathways. Biosynthesis of LA exclusively occurs in the apicoplast, comprising octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) and LA synthase. Salvage of LA is mitochondrial and scavenged LA is ligated to the KADHs by LA protein ligase 1 (LplA1). Both pathways are entirely independent, suggesting that both are likely to be essential for parasite survival. However, disruption of the LipB gene did not negatively affect parasite growth despite a drastic loss of LA (>90%). Surprisingly, the sole, apicoplast-located pyruvate dehydrogenase still showed Lipoylation, suggesting that an alternative Lipoylation pathway exists in this organelle. We provide evidence that this residual Lipoylation is attributable to the dual targeted, functional lipoate protein ligase 2 (LplA2). Localisation studies show that LplA2 is present in both mitochondrion and apicoplast suggesting redundancy between the lipoic acid protein ligases in the erythrocytic stages of P. falciparum.
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plasmodium falciparum possesses organelle specific α keto acid dehydrogenase complexes and Lipoylation pathways
Biochemical Society Transactions, 2005Co-Authors: Svenja Gunther, Lynsey J M Wallace, Paul J Mcmillan, Sylke MullerAbstract:The human malaria parasite Plasmodium falciparum possesses a single mitochondrion and a plastid-like organelle called the apicoplast. Both organelles contain members of the KADH (α-keto acid dehydrogenase) complexes – multienzyme complexes that are involved in intermediate metabolism. In the asexual blood stage forms of the parasites, the α-ketoglutarate dehydrogenase and branched chain KADH complexes are both located in the mitochondrion, whereas the pyruvate dehydrogenase is exclusively found in the apicoplast. In agreement with this distribution, Plasmodium parasites have two separate and organelle-specific pathways that guarantee Lipoylation of the KADH complexes in both organelles. A biosynthetic pathway comprised of lipoic acid synthase and lipoyl (octanoyl)-ACP:protein N e -lipoyltransferase B is present in the apicoplast, whereas the mitochondrion is supplied with exogenous lipoic acid, and ligation of the metabolite to the KADH complexes is accomplished by a lipoate protein ligase A similar to that of bacteria and plants. Both pathways are excellent potential targets for the design of new antimalarial drugs.
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the human malaria parasite plasmodium falciparum has distinct organelle specific Lipoylation pathways
Molecular Microbiology, 2004Co-Authors: Carsten Wrenger, Sylke MullerAbstract:Summary Lipoic acid is an essential cofactor of a -keto acid dehydrogenase complexes (KADHCs). This study shows that Plasmodium falciparum possesses two distinct Lipoylation pathways that are found in separate subcellular localizations. Lipoic acid synthesis comprising lipoic acid synthase and lipoyl-ACP:protein N -lipoyl transferase is present in the parasite’s apicoplast, whereas the second pathway consisting of lipoic acid protein ligase is located in the parasite’s mitochondrion. The two localizations were established by overexpressing green fluorescent protein fusions of the N-terminal sequences of lipoic acid synthase and lipoic acid protein ligase in intraerythrocytic stages of P. falciparum . Northern and Western blot analyses revealed that the genes/proteins encoding lipoic acid synthase, lipoyl-ACP:protein N -lipoyl transferase and lipoic acid protein ligase are expressed maximally in the early and late stages of P. falciparum erythrocytic development. The functionality of the three proteins was proven by complementation of bacteria deficient in lipA and lipB . Our results show that P. falciparum possesses two independent pathways, with different locations, responsible for the post-translational modification of KADHCs. Both pathways fundamentally differ from those in the human host. As KADHCs provide metabolites that are required for essential biosynthetic processes such as fatty acid biosynthesis and haem biosynthesis, the two Lipoylation pathways of P. falciparum might be attractive therapeutic targets against malaria.
Mareike G. Posner - 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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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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Comparative genomic analysis of Lipoylation pathways in archaea.
2014Co-Authors: Kirill Borziak, Michael J. Danson, Mareike G. Posner, Abhishek Upadhyay, Stefan Bagby, Steve DorusAbstract:The genomic presence of Lipoylation enzymes LplA-N, LipM or LipB and their substrate OADHC E2 is indicated. Archaeal orders lacking Lipoylation pathways are highlighted (grey shading). The broad metabolic environment of each archaeal order and the number of species analyzed are also indicated. Phylogenetic relationships are based on Brochier-Armanet et al. [53]; branch lengths are not drawn to scale.
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post translational modification in the archaea structural characterization of multi enzyme complex Lipoylation
Biochemical Journal, 2013Co-Authors: Mareike G. Posner, Michael J. Danson, Abhishek Upadhyay, Steve Dorus, Susan J Crennell, Andrew J Watson, Stefan BagbyAbstract:Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multi-enzyme complexes, is essential for metabolism in aerobic bacteria and eukarya. In Escherichia coli, Lipoylation is catalysed by LplA (lipoate protein ligase) or by LipA (lipoic acid synthetase) and LipB [lipoyl(octanoyl) transferase] combined. Whereas bacterial and eukaryotic LplAs comprise a single two-domain protein, archaeal LplA function typically involves two proteins, LplA-N and LplA-C. In the thermophilic archaeon Thermoplasma acidophilum, LplA-N and LplA-C are encoded by overlapping genes in inverted orientation (lpla-c is upstream of lpla-n). The T. acidophilum LplA-N structure is known, but the LplA-C structure is unknown and LplA-C's role in Lipoylation is unclear. In the present study, we have determined the structures of the substrate-free LplA-N-LplA-C complex and E2lipD (dihydrolipoyl acyltransferase lipoyl domain) that is lipoylated by LplA-N-LplA-C, and carried out biochemical analyses of this archaeal Lipoylation system. Our data reveal the following: (i) LplA-C is disordered but folds upon association with LplA-N; (ii) LplA-C induces a conformational change in LplA-N involving substantial shortening of a loop that could repress catalytic activity of isolated LplA-N; (iii) the adenylate-binding region of LplA-N-LplA-C includes two helices rather than the purely loop structure of varying order observed in other LplA structures; (iv) LplAN-LplA-C and E2lipD do not interact in the absence of substrate; (v) LplA-N-LplA-C undergoes a conformational change (the details of which are currently undetermined) during Lipoylation; and (vi) LplA-N-LplA-C can utilize octanoic acid as well as lipoic acid as substrate. The elucidated functional inter-dependence of LplA-N and LplA-C is consistent with their evolutionary co-retention in archaeal genomes.
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Lipoylation and assembly of a 2-oxoacid dehydrogenase multienzyme complex from thermoplasma acidophilum
2009Co-Authors: Mareike G. PosnerAbstract:Energy generating processes like the citric acid cycle are a pivotal part of metabolism. Members of the 2-oxoacid dehydrogenase multienzyme complex (OADHC) superfamily feed into and act within the citric acid cycle. OADHCs are composed of three enzymes: 2-oxoacid decarboxylase (E1), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). Covalent attachment of lipoic acid (LA) to E2 is essential for overall OADHC activity. Although thought to be absent in Archaea, it has recently been found that Thermoplasma acidophilum has all the components for an active recombinant OADHC (Heath et al., 2007). Recent studies have further suggested that Tp. acidophilum may have an enzyme to covalently attach LA to E2 (Sun et al., 2007; McManus et al., 2006). This work describes the cloning and recombinant expression of the Thermoplasma lipoate protein ligase (Tp. LplA), its C-terminal domain and a fusion protein composed of the above two proteins. Both proteins are required for Lipoylation of E2 in vitro. For the first time, in vivo Lipoylation of E2 in Tp. acidophilum cell cultures is also being reported. The effect of Lipoylation and temperature on the Thermoplasma OADHC assembly has also been studied. This study revealed the temperature dependence of the E2 core and the whole complex assembly. These findings are in line with the optimum growth temperature of Tp. acidophilum. Dynamic light scattering and analytical ultracentrifugation were used to determine the molecular mass of whole OADHC. The molecular mass was determined to be 5 MDa with an octahedral geometry of the E2 core. The results of this work strengthen the assumption that these enzyme systems may have had or potentially have a role in the Archaea. This may hold further clues to the evolutionary relationship between the three kingdoms of life and the role of OADHCs/Lipoylation in the Archaea. The temperature dependent assembly of the complex and thermostability of these proteins may also provide a model to study thermostability and protein-protein interactions at high temperatures.
Stuart Smith - One of the best experts on this subject based on the ideXlab platform.
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Compromised Mitochondrial Fatty Acid Synthesis in Transgenic Mice Results in Defective Protein Lipoylation and Energy Disequilibrium
2016Co-Authors: Stuart Smith, Andrzej Witkowski, Ayesha Moghul, Yuko Yoshinaga, Michael Nefedov, Dejiang Feng, Loren FongAbstract:A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational Lipoylation of pyruvate and a-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative Lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis o
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compromised mitochondrial fatty acid synthesis in transgenic mice results in defective protein Lipoylation and energy disequilibrium
PLOS ONE, 2012Co-Authors: Stuart Smith, Andrzej Witkowski, Ayesha Moghul, Yuko Yoshinaga, Michael Nefedov, Pieter J De Jong, Dejiang Feng, Loren G Fong, Stephen G Young, Thomas PhamAbstract:A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational Lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative Lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein Lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor.
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down regulation of mitochondrial acyl carrier protein in mammalian cells compromises protein Lipoylation and respiratory complex i and results in cell death
Journal of Biological Chemistry, 2009Co-Authors: Dejiang Feng, Andrzej Witkowski, Stuart SmithAbstract:The objective of this study was to evaluate the physiological importance of the mitochondrial fatty acid synthesis pathway in mammalian cells using the RNA interference strategy. Transfection of HEK293T cells with small interfering RNAs targeting the acyl carrier protein (ACP) component reduced ACP mRNA and protein levels by >85% within 24 h. The earliest phenotypic changes observed were a marked decrease in the proportion of post-translationally lipoylated mitochondrial proteins recognized by anti-lipoate antibodies and a reduction in their catalytic activity, and a slowing of the cell growth rate. Later effects observed included a reduction in the specific activity of respiratory complex I, lowered mitochondrial membrane potential, the development of cytoplasmic membrane blebs containing high levels of reactive oxygen species and ultimately, cell death. Supplementation of the culture medium with lipoic acid offered some protection against oxidative damage but did not reverse the protein Lipoylation defect. These observations are consistent with a dual role for ACP in mammalian mitochondrial function. First, as a key component of the mitochondrial fatty acid biosynthetic pathway, ACP plays an essential role in providing the octanoyl-ACP precursor required for the protein Lipoylation pathway. Second, as one of the subunits of complex I, ACP is required for the efficient functioning of the electron transport chain and maintenance of normal mitochondrial membrane potential.
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coupling of the de novo fatty acid biosynthesis and Lipoylation pathways in mammalian mitochondria
Journal of Biological Chemistry, 2007Co-Authors: Andrzej Witkowski, Anil K Joshi, Stuart SmithAbstract:The objective of this study was to identify the products and possible role of a putative pathway for de novo fatty acid synthesis in mammalian mitochondria. Bovine heart mitochondrial matrix preparations were prepared free from contamination by proteins from other subcellular components and, using a combination of radioisotopic labeling and mass spectrometry, were shown to contain all of the enzymes required for the extension of a 2-carbon precursor by malonyl moieties to saturated acyl-ACP thioesters containing up to 14 carbon atoms. A major product was octanoyl-ACP and, in the presence of the apo-H-protein of the glycine cleavage complex, the newly synthesized octanoyl moieties were translocated to the Lipoylation site on the acceptor protein. These studies demonstrate that one of the functions of the de novo fatty acid biosynthetic pathway in mammalian mitochondria is to provide the octanoyl precursor required for the essential protein Lipoylation pathway.
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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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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Comparative genomic analysis of Lipoylation pathways in archaea.
2014Co-Authors: Kirill Borziak, Michael J. Danson, Mareike G. Posner, Abhishek Upadhyay, Stefan Bagby, Steve DorusAbstract:The genomic presence of Lipoylation enzymes LplA-N, LipM or LipB and their substrate OADHC E2 is indicated. Archaeal orders lacking Lipoylation pathways are highlighted (grey shading). The broad metabolic environment of each archaeal order and the number of species analyzed are also indicated. Phylogenetic relationships are based on Brochier-Armanet et al. [53]; branch lengths are not drawn to scale.
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post translational modification in the archaea structural characterization of multi enzyme complex Lipoylation
Biochemical Journal, 2013Co-Authors: Mareike G. Posner, Michael J. Danson, Abhishek Upadhyay, Steve Dorus, Susan J Crennell, Andrew J Watson, Stefan BagbyAbstract:Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multi-enzyme complexes, is essential for metabolism in aerobic bacteria and eukarya. In Escherichia coli, Lipoylation is catalysed by LplA (lipoate protein ligase) or by LipA (lipoic acid synthetase) and LipB [lipoyl(octanoyl) transferase] combined. Whereas bacterial and eukaryotic LplAs comprise a single two-domain protein, archaeal LplA function typically involves two proteins, LplA-N and LplA-C. In the thermophilic archaeon Thermoplasma acidophilum, LplA-N and LplA-C are encoded by overlapping genes in inverted orientation (lpla-c is upstream of lpla-n). The T. acidophilum LplA-N structure is known, but the LplA-C structure is unknown and LplA-C's role in Lipoylation is unclear. In the present study, we have determined the structures of the substrate-free LplA-N-LplA-C complex and E2lipD (dihydrolipoyl acyltransferase lipoyl domain) that is lipoylated by LplA-N-LplA-C, and carried out biochemical analyses of this archaeal Lipoylation system. Our data reveal the following: (i) LplA-C is disordered but folds upon association with LplA-N; (ii) LplA-C induces a conformational change in LplA-N involving substantial shortening of a loop that could repress catalytic activity of isolated LplA-N; (iii) the adenylate-binding region of LplA-N-LplA-C includes two helices rather than the purely loop structure of varying order observed in other LplA structures; (iv) LplAN-LplA-C and E2lipD do not interact in the absence of substrate; (v) LplA-N-LplA-C undergoes a conformational change (the details of which are currently undetermined) during Lipoylation; and (vi) LplA-N-LplA-C can utilize octanoic acid as well as lipoic acid as substrate. The elucidated functional inter-dependence of LplA-N and LplA-C is consistent with their evolutionary co-retention in archaeal genomes.
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A unique Lipoylation system in the Archaea. Lipoylation in Thermoplasma acidophilum requires two proteins.
FEBS Journal, 2009Co-Authors: Mareike G. Posner, Abhishek Upadhyay, Stefan Bagby, Michael J. DansonAbstract:Members of the 2-oxoacid dehydrogenase multienzyme complex family play a key role in the pathways of central metabolism. Post-translational Lipoylation of the dihydrolipoyl acyltransferase component of these complexes is essential for their activity, the lipoyllysine moiety performing the transfer of substrates and intermediates between the different active sites within these multienzyme systems. We have previously shown that the thermophilic archaeon, Thermoplasma acidophilum, has a four-gene cluster encoding the components of such a complex, which, when recombinantly expressed in Escherichia coli, can be assembled into an active multienzyme in vitro. Crucially, the E. coli host carries out the required Lipoylation of the archaeal dihydrolipoyl acyltransferase component. Because active 2-oxoacid dehydrogenase multienzyme complexes have never been detected in any archaeon, the question arises as to whether Archaea possess a functional Lipoylation system. In this study, we report the cloning and heterologous expression of two genes from Tp. acidophilum whose protein products together show significant sequence identity with the single lipoate protein ligase enzyme of bacteria. We demonstrate that both recombinantly expressed Tp. acidophilum proteins are required for Lipoylation of the acyltransferase, and that the two proteins associate together to carry out this post-translational modification. From the published DNA sequences, we suggest the presence of functional transcriptional and translational regulatory elements, and furthermore we present preliminary evidence that Lipoylation occurs in vivo in Tp. acidophilum. This is the first report of the Lipoylation machinery in the Archaea, which is unique in that the catalytic activity is dependent on two separate gene products.