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PubMed1998

Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro.

Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli. The E2 component was purified as a large soluble aggregate (molecular mass > 1 x 10(6) Da) with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa. The recombinant E2 component in vitro was capable of binding either 60 E3(alpha2) dimers or 60 heterotetramers (alpha2beta2) of the pyruvate decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain. This has important consequences for the stoichiometry of the assembled complex in vivo. The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post-translationally modified in vitro using a recombinant lipoate protein ligase from E. coli. The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild-type enzyme. Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95% and 85%, respectively. However, only 26% of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1alpha and E1beta. This represents the first complete post-translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.

Genes, Bacterial↗
PubMed1997

A new metabolic link. The acyl carrier protein of lipid synthesis donates lipoic acid to the pyruvate dehydrogenase complex in Escherichia coli and mitochondria.

Lipoic acid is an essential enzyme cofactor that requires covalent attachment to its cognate proteins to confer biological activity. The major lipoylated proteins are highly conserved enzymes of central metabolism, the pyruvate and alpha-ketoglutarate dehydrogenase complexes. The classical lipoate ligase uses ATP to activate the lipoate carboxyl group followed by attachment of the cofactor to a specific subunit of each dehydrogenase complex, and it was assumed that all lipoate attachment proceeded by this mechanism. However, our previous work indicated that Escherichia coli could form lipoylated proteins in the absence of detectable ATP-dependent ligase activity raising the possibility of a class of enzyme that attaches lipoate to the dehydrogenase complexes by a different mechanism. We now report that E. coli and mitochondria contain lipoate transferases that use lipoyl-acyl carrier protein as the lipoate donor. This finding demonstrates a direct link between fatty acid synthesis and lipoate attachment and also provides the first direct demonstration of a role for the enigmatic acyl carrier proteins of mitochondria.

Acyl Carrier Protein↗
PubMed2004

Azorhizobium caulinodans electron-transferring flavoprotein N electrochemically couples pyruvate dehydrogenase complex activity to N2 fixation.

Azorhizobium caulinodans thermolabile point mutants unable to fix N2 at 42 degrees C were isolated and mapped to three, unlinked loci; from complementation tests, several mutants were assigned to the fixABCX locus. Of these, two independent fixB mutants carried missense substitutions in the product electron-transferring flavoprotein N (ETFN) alpha-subunit. Both thermolabile missense variants Y238H and D229G mapped to the ETFNalpha interdomain linker. Unlinked thermostable suppressors of these two fixB missense mutants were identified and mapped to the lpdA gene, encoding dihydrolipoamide dehydrogenase (LpDH), immediately distal to the pdhABC genes, which collectively encode the pyruvate dehydrogenase (PDH) complex. These two suppressor alleles encoded LpDH NAD-binding domain missense mutants G187S and E210G. Crude cell extracts of these fixB lpdA double mutants showed 60-70% of the wild-type PDH activity; neither fixB lpdA double mutant strain exhibited any growth phenotype at the restrictive or the permissive temperature. The genetic interaction between two combinations of lpdA and fixB missense alleles implies a physical interaction of their respective products, LpDH and ETFN. Presumably, this interaction electrochemically couples LpDH as the electron donor to ETFN as the electron acceptor, allowing PDH complex activity (pyruvate oxidation) to drive soluble electron transport via ETFN to N2, which acts as the terminal electron acceptor. If so, then, the A. caulinodans PDH complex activity sustains N2 fixation both as the driving force for oxidative phosphorylation and as the metabolic electron donor.

Azorhizobium caulinodans↗
PubMed1993

Halothane metabolism: the dihydrolipoamide acetyltransferase subunit of the pyruvate dehydrogenase complex molecularly mimics trifluoroacetyl-protein adducts.

Monospecific antibodies (anti-CF3CO antibodies), directed against trifluoroacetyl-protein adducts (CF3CO-protein adducts) that are elicited in tissues of experimental animals and humans upon exposure to the anesthetic agent halothane, recognize cross-reactive proteins of 64 and 52 kDa in several tissues of rats and the liver of humans not previously exposed to the drug. These cross-reactive proteins mimic CF3CO-protein adducts. Here, by the use of the anti-CF3CO antibody as an immunoaffinity matrix, the protein of 64 kDa was purified from rat heart microsomal fractions. The amino acid sequence of six internal tryptic peptides exhibited 100% identity with the corresponding deduced amino acid sequences of the dihydrolipoamide acetyltransferase component (E2 subunit) of the rat liver pyruvate dehydrogenase (PDH) complex, as encoded by the cDNA clone pRMIT [Gershwin, M. E., Mackay, I. R., Sturgess, A., & Coppel, R. L. (1987) J. Immunol. 138, 3525-3531]. Lipoic acid, the prosthetic group of the E2 subunit of the PDH complex, exhibited immunochemical properties very similar to those of the hapten-derivative N6-trifluoroacetyl-L-lysine (CF3CO-Lys). On immunoblots, free lipoic acid inhibited the recognition of the E2 subunit, of the not yet identified protein of 52 kDa, and of the bulk of CF3CO-protein adducts by anti-CF3CO antibody with half-maximal inhibitory constants of 0.05, 10.0, and 8.5 mM, respectively. Lipoic acid also abolished the precipitation of the native E2 subunit by anti-CF3CO antibody from solubilized rat heart mitochondrial fractions. These data suggest that lipoic acid is involved in the molecular mimicry of CF3CO-protein adduct-related epitopes by the E2 subunit of the PDH complex.

Acetyltransferases↗
PubMed2002

Identification of key amino acid residues in the assembly of enzymes into the pyruvate dehydrogenase complex of Bacillus stearothermophilus: a kinetic and thermodynamic analysis.

Structural studies have shown that electrostatic interactions play a major part in the binding of dihydrolipoyl dehydrogenase (E3) to the peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acyltransferase (E2) in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The binding is characterized by a small, unfavorable enthalpy change (deltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TdeltaS degrees = +14.8 kcal/mol). The contributions of individual surface residues of the PSBD of E2 to its interaction with E3 have been assessed by alanine-scanning mutagenesis, surface plasmon resonance detection, and isothermal titration calorimetry. The mutation R135A in the PSBD gave rise to a significant decrease (120-fold) in the binding affinity; two other mutations (R139A and R156A) were associated with smaller effects. The binding of the R135A mutant to E3 was accompanied by a favorable enthalpy (deltaH degrees = -2.6 kcal/mol) and a less positive entropy change (TdeltaS degrees = +7.2 kcal/mol). The midpoint melting temperature (T(m)) of E3-PSBD complexes was determined by differential scanning calorimetry. The R135A mutation caused a significant decrease (5 degrees C) in the T(m), compared with the wild-type complex. The results reveal the importance of Arg135 of the PSBD as a key residue in the molecular recognition of E3 by E2, and as a major participant in the overall entropy-driven binding process. Further, the effects of mutagenesis on the deltaCp of subunit association illustrate the difficulties in attributing changes in heat capacity to specific classes of interactions.

Acetyltransferases↗
PubMed1984

Activation of pyruvate dehydrogenase complex by porcine and biosynthetic human insulin in cultured human fibroblasts.

Cultured human fibroblasts represent an appropriate model for studying both insulin receptor interaction and hormone responsiveness. We have investigated the properties of the pyruvate dehydrogenase multi-enzyme complex (PDC) and have studied the effects of various concentrations of porcine and biosynthetic human insulin (BHI) on the activity of the enzyme. Under optimal conditions of the assay, both BHI and porcine insulin activated PDC in a dose-dependent fashion in which full activation of the enzyme was achieved with 10(-8) M insulin. The half-maximal concentration for porcine and human insulin was similar, occurring at the level of 5 X 10(-9) M for activation of the PDC of human fibroblasts. We conclude that the PDC of cultured human fibroblasts is activated by both human and porcine insulin at a comparable physiologic concentration. Human fibroblasts may therefore serve as a useful model to study insulin action in isolated human tissue.

Animals↗