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PubMed1983

The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the pyruvate dehydrogenase component.

The nucleotide sequence of a 3780-base-pair segment of DNA containing the aceE gene encoding the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The aceE structural gene comprises 2655 base pairs (885 codons, excluding the initiation codon AUG), it is preceded by a good ribosome binding site and several potential RNA polymerase binding sites. Its polarity and location in the restriction map of the corresponding segment of DNA are consistent with it being the proximal gene in the ace operon, as defined in previous genetic and post-infection labelling studies. The relative molecular mass (99474), composition (885 amino acids), amino-terminal residue and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with published information obtained from studies with the purified pyruvate dehydrogenase component (E1). The nucleotide sequence also contains a second gene (gene A) situated upstream of the aceE gene. It appears to be an independent gene containing 708 base pairs (236 codons) and encoding a weakly expressed product (protein A; Mr = 27049) of unknown function.

Amino Acids↗
PubMed1975

Activation of pyruvate dehydrogenase in adipose tissue by insulin. Evidence for an effect of insulin on pyruvate dehydrogenase phosphate phosphatase.

1. The mechanism by which insulin activates pyruvate dehydrogenase in rat epididymal adipose tissue was further investigated. 2. When crude extracts, prepared from tissue segments previously exposed to insulin (2m-i.u/ml) for 2min, were supplemented with Mg-2+, Ca-2+, glucose and hexokinase and incubated at 30 degrees C, they displayed an enhanced rate of increase in pyruvate dehydrogenase activity compared with control extracts. 3. When similar extracts were instead supplemented with fluoride, ADP, creatine phosphate and creatine kinase, the rate of decrease in pyruvate dehydrogenase activity observed during incubation at 30 degrees C was unaffected by insulin treatment. 4. It is suggested that insulin increases the fraction of pyruvate dehydrogenase present in the tissue in the active dephospho form by increasing the activity of pyruvate dehydrogenase phosphate phosphatase.

Adenosine Diphosphate↗
PubMed1996

Mutation analysis of the pyruvate dehydrogenase E1 alpha gene in eight patients with a pyruvate dehydrogenase complex deficiency.

Most of the mutations causing deficiency of the pyruvate dehydrogenase (PDH) complex are in the X-linked E1 alpha gene. We have developed a rapid screening method for the detection of mutations in this gene using reverse transcription of total RNA, polymerase chain reaction amplification of the whole coding region of the gene and single-strand conformation polymorphism (SSCP) analysis. With this method, we studied eight patients with a PDH complex deficiency, using cultured fibroblasts. In all patients, aberrant SSCP patterns were found and, after sequencing of the corresponding fragments, we were able to identify six new mutations and two mutations already described previously. The mutations are point mutations leading to amino acid substitutions (5) and direct repeat insertions (3). The presence of the mutations was confirmed in genomic fibroblast DNA. The 4 female patients were shown to carry both a normal and a mutated E1 alpha gene.

Base Sequence↗
PubMed2003

Biochemical and physiological studies of Arabidopsis thaliana transgenic lines with repressed expression of the mitochondrial pyruvate dehydrogenase kinase.

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. Previously, the cloning of a PDHK cDNA from Arabidopsis thaliana and the effects of constitutively down-regulating its expression on plant growth and development has been reported. The first detailed analyses of the biochemical and physiological effects of partial silencing of the mtPDHK in A. thaliana using antisense constructs driven by both constitutive and seed-specific promoters are reported here. The studies revealed an increased level of respiration in leaves of the constitutive antisense PDHK transgenics; an increase in respiration was also found in developing seeds of the seed-specific antisense transgenics. Both constitutive and seed-specific partial silencing of the mtPDHK resulted in increased seed oil content and seed weight at maturity. Feeding 3-(14)C pyruvate to bolted stems containing siliques (constitutive transgenics), or to isolated siliques or immature seeds (seed-specific transgenics) confirmed a higher rate of incorporation of radiolabel into all seed lipid species, particularly triacylglycerols. Neither constitutive nor seed-specific partial silencing of PDHK negatively affected overall silique and seed development. Instead, oil and seed yield, and overall plant productivity were improved. These findings suggest that a partial reduction of the repression of the mtPDC by antisense PDHK expression can alter carbon flux and, in particular, the contribution of carbon moieties from pyruvate to fatty acid biosynthesis and storage lipid accumulation in developing seeds, implicating a role for mtPDC in fatty acid biosynthesis in seeds.

Antisense Elements (Genetics)↗
PubMed2002

Identification of the E2-binding residues in the N-terminal domain of E1 of a prokaryotic pyruvate dehydrogenase complex.

Pyruvate dehydrogenase (E1p) is one of the components of the pyruvate dehydrogenase multienzyme complex (PDHC). Previously, it was shown that the N-terminal domain of E1p is involved in its binding to the core component (E2p) of PDHC. We constructed point mutations in this domain (D17Q, D17R, E20Q, E20R, D24Q and D24R) to identify the specific residues involved in these interactions. Kinetic and binding studies show that D17 is essential for the binding of E1p to E2p. D24 is involved in the binding, but not essential, whereas E20 is not involved. None of the mutations affects the folding or dimerisation of E1p.

Amino Acid Sequence↗
PubMed2005

How dihydrolipoamide dehydrogenase-binding protein binds dihydrolipoamide dehydrogenase in the human pyruvate dehydrogenase complex.

The dihydrolipoamide dehydrogenase-binding protein (E3BP) and the dihydrolipoamide acetyltransferase (E2) component enzyme form the structural core of the human pyruvate dehydrogenase complex by providing the binding sites for two other component proteins, dihydrolipoamide dehydrogenase (E3) and pyruvate dehydrogenase (E1), as well as pyruvate dehydrogenase kinases and phosphatases. Despite a high similarity between the primary structures of E3BP and E2, the E3-binding domain of human E3BP is highly specific to human E3, whereas the E1-binding domain of human E2 is highly specific to human E1. In this study, we characterized binding of human E3 to the E3-binding domain of E3BP by x-ray crystallography at 2.6-angstroms resolution, and we used this structural information to interpret the specificity for selective binding. Two subunits of E3 form a single recognition site for the E3-binding domain of E3BP through their hydrophobic interface. The hydrophobic residues Pro133, Pro154, and Ile157 in the E3-binding domain of E3BP insert themselves into the surface of both E3 polypeptide chains. Numerous ionic and hydrogen bonds between the residues of three interacting polypeptide chains adjacent to the central hydrophobic patch add to the stability of the subcomplex. The specificity of pairing for human E3BP with E3 is interpreted from its subcomplex structure to be most likely due to conformational rigidity of the binding fragment of the E3-binding domain of E3BP and its exquisite amino acid match with the E3 target interface.

Amino Acid Sequence↗
PubMed2002

Histidine mutagenesis of Arabidopsis thaliana pyruvate dehydrogenase kinase.

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Analysis of the primary amino-acid sequences of PDK from various sources reveals that these enzymes include the five domains characteristic of prokaryotic two-component His-kinases, despite the fact that PDK exclusively phosphorylates Ser residues in the E1alpha subunit of the PDC. This seeming contradiction might be resolved if the PDK-catalyzed reaction employed a phospho-His intermediate. The results from pH-stability studies of autophosphorylated Arabidopsis thaliana PDK did not provide any support for a phospho-His intermediate. Furthermore, site-directed mutagenesis of the two most likely phosphotransfer His residues (H121 and H168) did not abolish either PDK autophosphorylation or the ability to transphosphorylate E1alpha. Thus, PDK is a unique type of protein kinase having a His-kinase-like sequence but Ser-kinase activity.

Amino Acid Sequence↗
PubMed1998

Identification of the catalytic glutamate in the E1 component of human pyruvate dehydrogenase.

The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA. The first component (E1) converts pyruvate to bound acetaldehyde using thiamine diphosphate (ThDP) and Mg2+ as cofactors. There is no 3D structure of E1 available but those of other ThDP-dependent enzymes show some similarities including a glutamate residue that assists in ThDP activation. Eukaryotic E1 has an alpha2beta2 structure and the conserved Glu89 of the beta-subunit was identified as a possible catalytic residue by sequence alignment. Human E1 was expressed in Escherichia coli and purified. Mutating Glu89 to glutamine, aspartate and alanine markedly reduces catalytic activity and the affinity for ThDP, consistent with a role as the catalytic glutamate.

Amino Acid Sequence↗
PubMed1998

Gene and subunit organization of bacterial pyruvate dehydrogenase complexes.

Pyruvate dehydrogenase complexes of bacterial origin are compared with respect to subunit composition, organization of the corresponding genes, and the number and location of lipoyl domains. Special attention is given to two unusual examples of pyruvate dehydrogenase complexes, formed by Zymomonas mobilis and Thiobacillus ferrooxidans.

Bacteria↗
PubMed1981

Dihydrolipoamide dehydrogenase component of the pyruvate dehydrogenase complex from Escherichia coli K12. Comparative characterization of the free and the complex-bound component.

The regulation of the biosynthesis of dihydrolipoamide dehydrogenase is dependent on the biosynthesis of the pyruvate dehydrogenase complex. The gene coding for the dihydrolipoamide dehydrogenase appears to be included in the regulation of the pyruvate dehydrogenase operon. Possibly a secondary promoter is inserted. Dihydrolipoamide dehydrogenase was purified in its free form using a dihydrolipoamide-agarose affinity column and avoiding denaturating conditions. The enzyme shows complete cross-reactivity with antibodies against the pyruvate dehydrogenase complex and has a higher specific activity than any preparations described thus far. The transition state activation energy of the catalytic activity is smaller for the complex-bound enzyme than that found for the free enzyme. In its complexed form, the enzyme also proves to be somewhat more stable under alkaline conditions. The reaction catalyzed by the dihydrolipoamide dehydrogenase shows the behaviour of a ping-pong mechanism. The Michaelis constants for the substrates NAD and dihydrolipoamide found with the free enzyme are about four times those observed with the enzyme integrated into the native complex. The catalytic reaction of both forms of the dihydrolipoamide dehydrogenase is inhibited by NADH. The mechanism of this inhibition cannot simply be explained by a product inhibition. Rather the further reduction of the catalytically active, half-reduced enzyme form to the catalytically inactive, fully reduced form has to be considered as causing the inhibition.

Dihydrolipoamide Dehydrogenase↗
PubMed1991

The alpha-ketoacid dehydrogenase complexes. Sequence similarity of rat pyruvate dehydrogenase with Escherichia coli and Azotobacter vinelandii alpha-ketoglutarate dehydrogenase.

The pyruvate dehydrogenase complex and the alpha-ketoglutarate dehydrogenase complex are multienzyme complexes consisting of three different enzymes. No significant similarity has been reported among the dehydrogenases which are component enzymes of these complexes, despite the presence of homology among the other component enzymes. Here we isolated cDNAs for the alpha and beta subunits of rat pyruvate dehydrogenase and they exhibited a significant similarity of the amino acid sequences among rat pyruvate dehydrogenase, 2-oxoisovalerate dehydrogenase (which is a dehydrogenase component of branched chain alpha-ketoacid dehydrogenase complex) and alpha-ketoglutarate dehydrogenase, suggesting that they have been derived from a common ancestral dehydrogenase. Our results suggested that the alpha and beta subunits of the pyruvate and 2-oxoisovalerate dehydrogenases have been derived by the cleavage of the alpha-ketoglutarate dehydrogenase. However, we could not find significant homology between rat pyruvate dehydrogenase and Gram-negative bacterial pyruvate dehydrogenase.

Amino Acid Sequence↗
PubMed1995

Mutations in the X-linked E1 alpha subunit of pyruvate dehydrogenase: exon skipping, insertion of duplicate sequence, and missense mutations leading to the deficiency of the pyruvate dehydrogenase complex.

Human pyruvate dehydrogenase (PDH)-complex deficiency is an inborn error of metabolism that is extremely heterogeneous in its presentation and clinical course. In a study of 14 patients (7 females and 7 males), we have found a mutation in the coding region of the E1 alpha gene in all 14 patients. Two female patients had the same 7-bp deletion at nt 927; another female patient had a 3-bp deletion at nt 931. Another female patient was found to have a deletion of exon 6 in her cDNA. Two other female patients were found to have insertions, one of 13 bp at nt 981 and one of 46 bp at nucleotide 1078. Two male patients were found to have a 4-bp insertion at nucleotide 1163. The remaining six patients all had missense mutations. A male patient and a female patient both had an A1133G mutation. The other missense mutations were C214T, C615A, and C787G (two patients). Five of these mutations are novel mutations, five have been previously reported in other patients, and two were published observations in other patients in an E1 alpha-mutation summary. In the four cases where parent DNA was available, only one mother was found to be a carrier of the same mutation as her child.

Base Sequence↗
PubMed1997

In situ nucleic acid hybridization of pyruvate dehydrogenase complex-E2 in primary biliary cirrhosis: pyruvate dehydrogenase complex-E2 messenger RNA is expressed in hepatocytes but not in biliary epithelium.

Pyruvate dehydrogenase-E2, or a cross-reactive molecule, has been shown by a variety of immunohistochemical methods to be present in increased amounts in biliary epithelial cells (BEC) in primary biliary cirrhosis (PBC). In this study, to further understand the nature of the immunoreactive molecule in BEC, we examined the expression of pyruvate dehydrogenase complex-E2 (PDC-E2) messenger RNA (mRNA) and PDC-E2 protein in sections of livers from patients and controls to help identify the molecule found in BEC. We performed in situ hybridization using an antisense probe against the major epitope of PDC-E2. The data were very striking and suggested that there was no increased production of PDC-E2 in BEC. For example, in livers from patients with PBC, PDC-E2 mRNA was found in periportal hepatocytes in 16 of 17 cases (94%). In contrast, interlobular bile ducts and septal bile ducts had detectable levels of PDC-E2 mRNA in only 1 of 17 (6%) and 3 of 8 (38%) cases, respectively. Interestingly, proliferating bile ductules contained detectable levels of mRNA in 12 of 15 cases (80%). In control liver, periportal hepatocytes were positive in 15 of 17 cases (88%). Interlobular bile ducts, septal bile ducts, and proliferating bile ductules expressed mRNA signals in 4 of 17 (24%), 2 of 10 (20%), and 14 of 16 (88%), respectively. When formalin-fixed, paraffin-embedded sections were examined by immunohistochemical staining with anti-PDC-E2 monoclonal antibody (mAb) C355.1, the interlobular bile ducts showed typical aberrant apical staining in all 10 PBC cases, but 0 of 9 liver controls. Periportal hepatocytes, proliferating bile ductules and infiltrating mononuclear cells stained with C355.1 but in a characteristic mitochondrial staining pattern. The presence of a PDC-E2-like molecule recognized by C355.1 is not reflected by the expression levels of PDC-E2 mRNA in the BEC of patients with PBC.

Bile Ducts↗
PubMed1989

Subunit associations in the mammalian pyruvate dehydrogenase complex. Structure and role of protein X and the pyruvate dehydrogenase component binding domain of the dihydrolipoyl transacetylase component.

We have further distinguished the structures and roles of the two lipoyl-bearing components of the pyruvate dehydrogenase complex, the dihydrolipoyl transacetylase (E2) component and the component designated as protein X. The amino acid sequences of the NH2-terminal regions of the lipoyl-bearing domain of the E2 component and protein X are different but related. The dihydrolipoyl dehydrogenase (E3) component but not the pyruvate dehydrogenase (E1) component protected protein X against proteolytic degradation by trypsin and protease Arg C. Protein X-specific polyclonal antibodies inhibit reconstitution of the overall reaction catalyzed by the complex (E2-X subcomplex recombined with the E1 and E3 components). The rate of development of this inhibition was reduced by pretreatment of E2-X subcomplex with the E3 component. These data strongly suggest the E3 component associates with protein X. The E1 component (an alpha 2 beta 2 tetramer), but not the E3 component, reduced trypsin cleavage of E2 subunits at 4 degrees C and altered the patterns of cleavage at 22 degrees C. At 22 degrees C a large (Mr congruent to 49,000) outer domain (E2LB) of the E2 component was produced. E2LB had the same NH2-terminal amino acid sequence as the smaller (Mr congruent to 38,000) lipoyl-bearing domain (E2L). E2LB, in contrast to E2L, interacted with both the E1 component and the beta subunit of the E1 component. Thus the E1 component is bound through an E1-binding domain that is located in E2 subunits between the inner domain and the outer, lipoyl-bearing domain.

Acetyltransferases↗