The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus: preparation and characterization of its binding to the dihydrolipoyl dehydrogenase component.
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ABSTRACT:
SUBMITTER: Hipps DS
PROVIDER: S-EPMC1137802 | biostudies-other | 1994 Jan
REPOSITORIES: biostudies-other
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Project description:The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus comprises a structural core, composed of 60 dihydrolipoamide acetyltransferase (E2p) subunits, which binds multiple copies of pyruvate decarboxylase (E1p) and dihydrolipoamide dehydrogenase (E3) subunits. After limited proteolysis with chymotrypsin, the N-terminal lipoyl domain of E2p was excised, purified and sequenced. The residual complex, which remained assembled, was then digested with trypsin under mild conditions. This treatment promoted complete disassembly of the complex and the various components were separated by gel filtration and h.p.l.c. A folded fragment of E2p containing about 50 amino acid residues was identified as being responsible for binding the E3 subunits, although, unlike the corresponding region of the E2p or E2o chains of the pyruvate dehydrogenase or 2-oxoglutarate dehydrogenase complexes from Escherichia coli, the fragment also bound E1p molecules. Further peptide purification and sequence analysis allowed the determination of the first 211 amino acid residues of the B. stearothermophilus E2p chain, thus providing the complete primary structure of the lipoyl domain, the E1p/E3-binding domain and the regions of polypeptide chain, probably highly flexible in nature, that link the domains to each other and to the inner-core (E2p-binding) domain. Several of the proteolytically sensitive sites were also identified. The sequence of the B. stearothermophilus E2p chain shows close homology with the sequences of the E2p and E2o chains from E. coli, although significant differences in structure are apparent. Detailed evidence for the sequence of the peptides obtained by limited proteolysis and further chemical and enzymic cleavages have been deposited as Supplementary Publication SUP 50142 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 6BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5.
| S-EPMC1149109 | biostudies-other
Project description:A sub-gene encoding the N-terminal 170 residues of the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. The expressed polypeptide consists of the lipoyl domain, inter-domain linker and peripheral subunit-binding domain; these were found to have folded into their native functional conformations as judged by reductive acetylation of the lipoyl domain, limited proteolysis of the linker region and ability to bind the dihydrolipoamide dehydrogenase dimer. The di-domain was largely (80%) unlipoylated; a small proportion (4%) was correctly modified with lipoic acid and the remainder (16%) was aberrantly modified with octanoic acid. A polyclonal antiserum was raised that recognized both the di-domain and the individual component domains. The 400 MHz 1H-n.m.r. spectrum of the di-domain showed resonances corresponding to those seen in spectra of the lipoyl domain, plus others characteristic of amino acid residues in the flexible linker region. Further, as yet unidentified, resonances are likely to be derived from the peripheral subunit-binding domain. The existence and independent folding of the peripheral subunit-binding domain is thus confirmed and its purification in large-scale amounts for detailed structural analysis is now possible.
| S-EPMC1130937 | biostudies-other
Project description:The interaction between the pyruvate decarboxylase (E1) component and a di-domain (lipoyl domain plus peripheral subunit-binding domain) from the dihydrolipoyl acetyltransferase (E2) component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex was investigated. Only 1 mol of di-domain (binding domain) was bound to 1 mol of heterotetrameric E1 (alpha 2 beta 2) and the binding was without effect on the kinetic activity of E1. Similarly, the di-domain bound to separate E1 beta subunits at a maximal polypeptide chain ratio of 1:2, but no detectable interaction was found with the E1 alpha subunit. However, addition of the monomeric E1 alpha subunit to an E1 beta-di-domain complex generated a fully functional E1 (alpha 2 beta 2)-di-domain complex, indicating that the E1 beta subunit plays the critical part in binding the E1 component to the di-domain and suggesting that no chaperonin is needed in vitro to promote the assembly of the three separate proteins. Mixing the E1 and dihydrolipoyl dehydrogenase (E3) components in the presence of di-domain revealed that E1 and E3 cannot bind simultaneously to the same molecule of di-domain, a new feature of the assembly pathway and an important factor in determining the ultimate structure of the assembled enzyme complex.
| S-EPMC1136582 | biostudies-other
Project description:1. The pyruvate dehydrogenase complex was purified from Bacillus stearothermophilus in high yield. The specific activity (about 40nkat/mg of protein) was substantially lower than that of the pyruvate dehydrogenase complex from Escherchia coli (about 570nkat/mg of protein) measured at 30 degrees C under the same conditions. 2. The relative molecular masses of the four types of polypeptide chain i the complex were estimated by means of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to be 57 000, 54 000, 42 000 and 36 000 respectively. These polypetide chains showed no evidence of seriously anomalous behavior during tests of electrophoretic mobility. 3. The enzyme complex was resolved into its constituent proteins by means of gelfiltration on Sepharose CL-6B in the presence of 2M-KI, followed by chromatography on hydroxyapatite in the presence of 8M-urea. These harsh conditions were necessary to cause suitable dissociation of the enzyme complex. 4. The amino-acid compositions of the four constituent proteins after resolution were determined and their chain ratios were measured for several preparations of the complex. Some variability was noted between preparations but all samples contained a significant molar excess of the chains thought to contribute the pyruvate decarboxylase (EC 1.2.4.1) activity. 5. From the relative molecular masses and chain ratios of the four constituent proteins, it was calculated that the empirical unit must be repeated at least 50 times to make up the assembled complex. This conclusion is fully consistent with the demonstration by means of electron microscopy of apparent icosahedral symmetry for the Bacillus stearothermophilus complex, implying a 60-fold repeat. The structure stands in sharp contrast with the octahedral symmetry (24-fold repeat) of the Escherichia coli enzyme.
| S-EPMC1161928 | biostudies-other
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).
| S-EPMC2676041 | biostudies-literature
Project description:The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s(-1), comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4'-aminopyrimidine tautomer of bound thiamin diphosphate (AP).
| S-EPMC4140881 | biostudies-literature
Project description:The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly.
| S-EPMC4208021 | biostudies-literature
Project description:The protein pirin, which is involved in a variety of biological processes, is conserved from prokaryotic microorganisms, fungi, and plants to mammals. It acts as a transcriptional cofactor or an apoptosis-related protein in mammals and is involved in seed germination and seedling development in plants. In prokaryotes, while pirin is stress induced in cyanobacteria and may act as a quercetinase in Escherichia coli, the functions of pirin orthologs remain mostly uncharacterized. We show that the Serratia marcescens pirin (pirin(Sm)) gene encodes an ortholog of pirin protein. Protein pull-down and bacterial two-hybrid assays followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization-tandem mass spectrometry analyses showed the pyruvate dehydrogenase (PDH) E1 subunit as a component interacting with the pirin(Sm) gene. Functional analyses showed that both PDH E1 subunit activity and PDH enzyme complex activity are inhibited by the pirin(Sm) gene in S. marcescens CH-1. The S. marcescens CH-1 pirin(Sm) gene was subsequently mutated by insertion-deletion homologous recombination. Accordingly, the PDH E1 and PDH enzyme complex activities and cellular ATP concentration increased up to 250%, 140%, and 220%, respectively, in the S. marcescens CH-1 pirin(Sm) mutant. Concomitantly, the cellular NADH/NAD(+) ratio increased in the pirin(Sm) mutant, indicating increased tricarboxylic acid (TCA) cycle activity. Our results show that the pirin(Sm) gene plays a regulatory role in the process of pyruvate catabolism to acetyl coenzyme A through interaction with the PDH E1 subunit and inhibiting PDH enzyme complex activity in S. marcescens CH-1, and they suggest that pirin(Sm) is an important protein involved in determining the direction of pyruvate metabolism towards either the TCA cycle or the fermentation pathways.
| S-EPMC1797226 | biostudies-literature
Project description:The pyruvate dehydrogenase complex of Bacillus stearothermophilus was treated with Staphylococcus aureus V8 proteinase, causing cleavage of the dihydrolipoamide acetyltransferase polypeptide chain (apparent Mr 57 000), inhibition of the enzymic activity and disassembly of the complex. Fragments of the dihydrolipoamide acetyltransferase chains with apparent Mr 28 000, which contained the acetyltransferase activity, remained assembled as a particle ascribed the role of an inner core of the complex. The lipoic acid residue of each dihydrolipoamide acetyltransferase chain was found as part of a small but stable domain that, unlike free lipoamide, was able still to function as a substrate for reductive acetylation by pyruvate in the presence of intact enzyme complex or isolated pyruvate dehydrogenase (lipoamide) component. The lipoyl domain was acidic and had an apparent Mr of 6500 (by sedimentation equilibrium), 7800 (by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) and 10 000 and 20 400 (by gel filtration in the presence and in the absence respectively of 6M-guanidinium chloride). 1H-n.m.r. spectroscopy of the dihydrolipoamide acetyltransferase inner core demonstrated that it did not contain the segments of highly mobile polypeptide chain found in the pyruvate dehydrogenase complex. 1H-n.m.r. spectroscopy of the lipoyl domain demonstrated that it had a stable and defined tertiary structure. From these and other experiments, a model of the dihydrolipoamide acetyltransferase chain is proposed in which the small, folded, lipoyl domain comprises the N-terminal region, and the large, folded, core-forming domain that contains the acetyltransferase active site comprises the C-terminal region. These two regions are separated by a third segment of the chain, which includes a substantial region of polypeptide chain that enjoys high conformational mobility and facilitates movement of the lipoyl domain between the various active sites in the enzyme complex.
| S-EPMC1153199 | biostudies-other
Project description:Th17 cells favor glycolytic metabolism, and pyruvate dehydrogenase (PDH) is the key bifurcation enzyme, which in its active dephosphorylated form advances the oxidative phosphorylation from glycolytic pathway. The transcriptional factor, inducible cAMP early repressor/cAMP response element modulator (ICER/CREM), has been shown to be induced in Th17 cells and to be overexpressed in CD4+ T cells from the patients with systemic lupus erythematosus (SLE). We found that glycolysis and lactate production in in vitro Th17-polarized T cells was reduced and that the expression of pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2), an enzyme that converts the inactive PDH to its active form, and PDH enzyme activity were increased in Th17 cells from ICER/CREM-deficient animals. ICER was found to bind to the Pdp2 promoter and suppress its expression. Furthermore, forced expression of PDP2 in CD4+ cells reduced the in vitro Th17 differentiation, whereas shRNA-based suppression of PDP2 expression increased in vitro Th17 differentiation and augmented experimental autoimmune encephalomyelitis. At the translational level, PDP2 expression was decreased in memory Th17 cells from patients with SLE and forced expression of PDP2 in CD4+ T cells from lupus-prone MRL/lpr mice and patients with SLE suppressed Th17 differentiation. These data demonstrate the direct control of energy production during Th17 differentiation in health and disease by the transcription factor ICER/CREM at the PDH metabolism bifurcation level.
| S-EPMC6140513 | biostudies-literature