Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato.
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ABSTRACT:
SUBMITTER: Millar AH
PROVIDER: S-EPMC1219725 | biostudies-other | 1998 Sep
REPOSITORIES: biostudies-other
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Project description:The 2-oxoglutarate dehydrogenase complex (OGDC) in potato (Solanum tuberosum cv. Romano) tuber mitochondria is largely associated with the membrane fraction of osmotically ruptured organelles, whereas most of the other tricarboxylic acid cycle enzymes are found in the soluble matrix fraction. The purification of OGDC from either membrane or soluble matrix fractions resulted in the increasing dependence of its activity on the addition of dihydrolipoamide dehydrogenase (E3). A 30-fold purification of OGDC to apparent homogeneity and with a specific activity of 4.6 micromol/min per mg of protein in the presence of exogenously added E3 was obtained. SDS/PAGE revealed that the purified complex consisted of three major polypeptides with apparent molecular masses of 48, 50 and 105 kDa. Before the gel-filtration purification step, E3 polypeptides of 57 and 58 kDa were identified by immunoreaction as minor proteins associated with OGDC. The N-terminal sequence of the 57 kDa protein was identical with that previously purified as the E3 component of the pyruvate dehydrogenase complex from potato. The 105 kDa protein was identified as the 2-oxoglutarate dehydrogenase subunit of OGDC by N-terminal sequencing. The N-terminal sequences of the 50 and 48 kDa proteins shared 90-95% identity over 20 residues and were identified by sequence similarity as dihydrolipoamide succinyltransferases (OGDC-E2). The incubation of OGDC with [U-(14)C]2-oxoglutarate resulted in the reversible succinylation of both the 48 and the 50 kDa protein bands. Proteins previously reported as subunits of complex I of the respiratory chain from Vicia faba and Solanum tuberosum are proposed to be OGDC-E2 and the possible basis of this association is discussed.
| S-EPMC1220557 | biostudies-other
Project description:Congenital lactic acidosis due to pyruvate dehydrogenase phosphatase (PDP) deficiency is very rare. PDP regulates pyruvate dehydrogenase complex (PDC) and defective PDP leads to PDC deficiency. We report a case with functional PDC deficiency with low activated (+dichloroacetate) and inactivated (+fluoride) PDC activities in lymphocytes and fibroblasts, normal activity of other mitochondrial enzymes in fibroblasts, and novel biallelic frameshift mutation in the PDP1 gene, c.575dupT (p.L192FfsX5), with absent PDP1 product in fibroblasts. Unexpectedly, the patient also had low branched-chain 2-ketoacid dehydrogenase (BCKDH) activity in fibroblasts with slight elevation of branched-chain amino acids in plasma and ketoacids in urine but with no pathogenic mutations in the enzymes of BCKDH, which could suggest shared regulatory function of PDC and BCKDH in fibroblasts, potentially in other tissues or cell types as well, but this remains to be determined. The clinical presentation of this patient overlaps that of other patients with primary-specific PDC deficiency, with neonatal/infantile and childhood lactic acidosis, normal lactate to pyruvate ratio, elevated plasma alanine, delayed psychomotor development, epileptic encephalopathy, feeding difficulties, and hypotonia. This patient exhibited marked improvement of overall development following initiation of ketogenic diet at 31 months of age. To the best of our knowledge, this is the fourth case of functional PDC deficiency with a defined mutation in PDP1.SynopsisPyruvate dehydrogenase phosphatase (PDP) regulates pyruvate dehydrogenase complex (PDC) and defective PDP due to PDP1 mutations leads to PDC deficiency and congenital lactic acidosis.
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Project description:The pyruvate dehydrogenase (PDH) complex of the gram-negative bacterium Zymomonas mobilis was purified to homogeneity. From 250 g of cells, we isolated 1 mg of PDH complex with a specific activity of 12.6 U/mg of protein. Analysis of subunit composition revealed a PDH (E1) consisting of the two subunits E1alpha (38 kDa) and E1beta (56 kDa), a dihydrolipoamide acetyltransferase (E2) of 48 kDa, and a lipoamide dehydrogenase (E3) of 50 kDa. The E2 core of the complex is arranged to form a pentagonal dodecahedron, as shown by electron microscopic images, resembling the quaternary structures of PDH complexes from gram-positive bacteria and eukaryotes. The PDH complex-encoding genes were identified by hybridization experiments and sequence analysis in two separate gene regions in the genome of Z. mobilis. The genes pdhAalpha (1,065 bp) and pdhAbeta (1,389 bp), encoding the E1alpha and E1beta subunits of the E1 component, were located downstream of the gene encoding enolase. The pdhB (1,323 bp) and lpd (1,401 bp) genes, encoding the E2 and E3 components, were identified in an unrelated gene region together with a 450-bp open reading frame (ORF) of unknown function in the order pdhB-ORF2-lpd. Highest similarities of the gene products of the pdhAalpha, pdhAbeta, and pdhB genes were found with the corresponding enzymes of Saccharomyces cerevisiae and other eukaryotes. Like the dihydrolipoamide acetyltransferases of S. cerevisiae and numerous other organisms, the product of the pdhB gene contains a single lipoyl domain. The E1beta subunit PDH was found to contain an amino-terminal lipoyl domain, a property which is unique among PDHs.
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Project description:Electron cryo-microscopy of 'single particles' is a powerful method to determine the three-dimensional (3D) architectures of complex cellular assemblies. The pyruvate dehydrogenase multi-enzyme complex couples the activity of three component enzymes (E1, E2 and E3) in the oxidative decarboxylation of pyruvate to generate acetyl-CoA, linking glycolysis and the tricarboxylic acid cycle. We report here a 3D model for an 11 MDa, icosahedral pyruvate dehydrogenase sub-complex, obtained by combining a 28 A structure derived from electron cryo-microscopy with previously determined atomic coordinates of the individual E1 and E2 components. A key feature is that the E1 molecules are located on the periphery of the assembly in an orientation that allows each of the 60 mobile lipoyl domains tethered to the inner E2 core to access multiple E1 and E2 active sites from inside the icosahedral complex. This unexpected architecture provides a highly efficient mechanism for active site coupling and catalytic rate enhancement by the motion of the lipoyl domains in the restricted annular region between the inner core and outer shell of the complex.
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Project description:The human pyruvate dehydrogenase complex (PDC) is a 9.5-megadalton catalytic machine that employs three catalytic components, i.e. pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), to carry out the oxidative decarboxylation of pyruvate. The human PDC is organized around a 60-meric dodecahedral core comprising the C-terminal domains of E2p and a noncatalytic component, E3-binding protein (E3BP), which specifically tethers E3 dimers to the PDC. A central issue concerning the PDC structure is the subunit stoichiometry of the E2p/E3BP core; recent studies have suggested that the core is composed of 48 copies of E2p and 12 copies of E3BP. Here, using an in vitro reconstituted PDC, we provide densitometry, isothermal titration calorimetry, and analytical ultracentrifugation evidence that there are 40 copies of E2p and 20 copies of E3BP in the E2p/E3BP core. Reconstitution with saturating concentrations of E1p and E3 demonstrated 40 copies of E1p heterotetramers and 20 copies of E3 dimers associated with the E2p/E3BP core. To corroborate the 40/20 model of this core, the stoichiometries of E3 and E1p binding to their respective binding domains were reexamined. In these binding studies, the stoichiometries were found to be 1:1, supporting the 40/20 model of the core. The overall maximal stoichiometry of this in vitro assembled PDC for E2p:E3BP:E1p:E3 is 40:20:40:20. These findings contrast a previous report that implicated that two E3-binding domains of E3BP bind simultaneously to a single E3 dimer (Smolle, M., Prior, A. E., Brown, A. E., Cooper, A., Byron, O., and Lindsay, J. G. (2006) J. Biol. Chem. 281, 19772-19780).
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Project description:The human (h) pyruvate dehydrogenase complex (hPDC) consists of multiple copies of several components: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3), E3-binding protein (BP), and specific kinases and phosphatases. Mammalian PDC has a well organized structure with an icosahedral symmetry of the central E2/BP core to which the other component proteins bind non-covalently. Both hE2 and hBP consist of three well defined domains, namely the lipoyl domain, the subunit-binding domain and the inner domain, connected with flexible linkers. hE1 (alpha(2)beta(2)) binds to the subunit-binding domain of hE2; whereas hE3 binds to the E3-binding domain of hBP. Among several residues of the C-terminal surface of the hE1beta E1betaD289 was found to interact with hE2K276. The C-terminal residue I329 of the hE1beta did not participate in binding to hE2. This latter finding shows specificity in the interaction between E1beta and E2 in hPDC. The selective binding between hE3 and the E3-binding domain of hBP was investigated using specific mutants. E3R460G and E3340K showed significant reductions in affinity for hBP as determined by surface plasmon resonance. Both residues are involved in the structural organization of the binding site on hE3. Substitution of I157, N137 and R155 of hBP resulted in variable increases in the K(D) for binding with wild-type hE3, suggesting that the binding results from several weak electrostatic bonds and hydrophobic interactions among residues of hBP with residues at the interface of dimeric hE3. These results provide insight in the mono-specificity of binding of E1 to E2 and E3 to BP in hPDC and showed the differences in the binding of peripheral components (E1 and E3) in human and bacterial PDCs.
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