scholarly journals Antimitochondrial Antibody Recognition and Structural Integrity of the Inner Lipoyl Domain of the E2 Subunit of Pyruvate Dehydrogenase Complex

2013 ◽  
Vol 191 (5) ◽  
pp. 2126-2133 ◽  
Author(s):  
Jinjun Wang ◽  
Madhu S. Budamagunta ◽  
John C. Voss ◽  
Mark J. Kurth ◽  
Kit S. Lam ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Structural Integrity ◽  
Pyruvate Dehydrogenase Complex ◽  
Antibody Recognition ◽  
Antimitochondrial Antibody ◽  
Inner Lipoyl Domain ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

scholarly journals Expression and lipoylation in Escherichia coli of the inner lipoyl domain of the E2 component of the human pyruvate dehydrogenase complex

1993 ◽  
Vol 289 (1) ◽  
pp. 81-85 ◽  
Author(s):  
J Quinn ◽  
A G Diamond ◽  
A K Masters ◽  
D E Brookfield ◽  
N G Wallis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Structural Studies ◽  
Gene Encoding ◽  
E Coli ◽  
Inner Lipoyl Domain ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The dihydrolipoamide acetyltransferase subunit (E2p) of mammalian pyruvate dehydrogenase complex has two highly conserved lipoyl domains each modified with a lipoyl cofactor bound in amide linkage to a specific lysine residue. A sub-gene encoding the inner lipoyl domain of human E2p has been over-expressed in Escherichia coli. Two forms of the domain have been purified, corresponding to lipoylated and non-lipoylated species. The apo-domain can be lipoylated in vitro with partially purified E. coli lipoate protein ligase, and the lipoylated domain can be reductively acetylated by human E1p (pyruvate dehydrogenase). Availability of the two forms will now allow detailed biochemical and structural studies of the human lipoyl domains.

scholarly journals Versatile Metabolic Adaptations ofRalstonia eutrophaH16 to a Loss of PdhL, the E3 Component of the Pyruvate Dehydrogenase Complex

2011 ◽  
Vol 77 (7) ◽  
pp. 2254-2263 ◽  
Author(s):  
Matthias Raberg ◽  
Jan Bechmann ◽  
Ulrike Brandt ◽  
Jonas Schlüter ◽  
Bianca Uischner ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Burkholderia Cepacia ◽  
Ralstonia Eutropha ◽  
Pyruvate Dehydrogenase Complex ◽  
Synthesis Rate ◽  
Wild Type ◽  
Dimerization Domain ◽  
Amino Terminal ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

ABSTRACTA previous study reported that the Tn5-induced poly(3-hydroxybutyric acid) (PHB)-leaky mutantRalstonia eutrophaH1482 showed a reduced PHB synthesis rate and significantly lower dihydrolipoamide dehydrogenase (DHLDH) activity than the wild-typeR. eutrophaH16 but similar growth behavior. Insertion of Tn5was localized in thepdhLgene encoding the DHLDH (E3 component) of the pyruvate dehydrogenase complex (PDHC). Taking advantage of the available genome sequence ofR. eutrophaH16, observations were verified and further detailed analyses and experiments were done.In silicogenome analysis revealed thatR. eutrophapossesses all five known types of 2-oxoacid multienzyme complexes and five DHLDH-coding genes. Of these DHLDHs, only PdhL harbors an amino-terminal lipoyl domain. Furthermore, insertion of Tn5inpdhLof mutant H1482 disrupted the carboxy-terminal dimerization domain, thereby causing synthesis of a truncated PdhL lacking this essential region, obviously leading to an inactive enzyme. The defined ΔpdhLdeletion mutant ofR. eutrophaexhibited the same phenotype as the Tn5mutant H1482; this excludes polar effects as the cause of the phenotype of the Tn5mutant H1482. However, insertion of Tn5or deletion ofpdhLdecreases DHLDH activity, probably negatively affecting PDHC activity, causing the mutant phenotype. Moreover, complementation experiments showed that different plasmid-encoded E3 components ofR. eutrophaH16 or of other bacteria, likeBurkholderia cepacia, were able to restore the wild-type phenotype at least partially. Interestingly, the E3 component ofB. cepaciapossesses an amino-terminal lipoyl domain, like the wild-type H16. A comparison of the proteomes of the wild-type H16 and of the mutant H1482 revealed striking differences and allowed us to reconstruct at least partially the impressive adaptations ofR. eutrophaH1482 to the loss of PdhL on the cellular level.

scholarly journals Cross-linking and 1H n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Escherichia coli

1982 ◽  
Vol 205 (2) ◽  
pp. 389-396 ◽  
Author(s):  
Leonard C. Packman ◽  
Richard N. Perham ◽  
Gordon C. K. Roberts
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
The Other ◽  
Random Coil ◽  
Cross Linking ◽  
Enzyme Complex ◽  
Cross Links ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The pyruvate dehydrogenase complex of Escherichia coli was treated with o-phenylene bismaleimide in the presence of the substrate pyruvate, producing almost complete cross-linking of the lipoate acetyltransferase polypeptide chains as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This took place without effect on the catalytic activities of the other two component enzymes and with little evidence of cross-links being formed with other types of protein subunit. Limited proteolysis with trypsin indicated that the cross-links were largely confined to the lipoyl domains of the lipoate acetyltransferase component of the same enzyme particle. This intramolecular cross-linking had no effect on the very sharp resonances observed in the 1H n.m.r. spectrum of the enzyme complex, which derive from regions of highly mobile polypeptide chain in the lipoyl domains. Comparison of the spin–spin relaxation times, T2, with the measured linewidths supported the idea that the highly mobile region is best characterized as a random coil. Intensity measurements in spin-echo spectra showed that it comprises a significant proportion (probably not less than one-third) of a lipoyl domain and is thus much more than a small hinge region, but there was insufficient intensity in the resonances to account for the whole lipoyl domain. On the other hand, no evidence was found in the 1H n.m.r. spectrum for a substantial structured region around the lipoyl-lysine residues that was free to move on the end of this highly flexible connection. If such a structured region were bound to other parts of the enzyme complex for a major part of its time, its resonances might be broadened sufficiently to evade detection by 1H n.m.r. spectroscopy.

scholarly journals Heterologously expressed inner lipoyl domain of dihydrolipoyl acetyltransferase inhibits ATP-dependent inactivation of pyruvate dehydrogenase complex

1998 ◽  
Vol 334 (3) ◽  
pp. 703-711 ◽  
Author(s):  
John C. JACKSON ◽  
Christine C. VINLUAN ◽  
Carol J. DRAGLAND ◽  
Vijayayakumar SUNDARARAJAN ◽  
Bing YAN ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Catalytic Subunits ◽  
Dependent Inactivation ◽  
Dihydrolipoyl Acetyltransferase ◽  
Animal Models Of Diabetes ◽  
Inner Lipoyl Domain ◽  
Lipoyl Domain

The activity of the pyruvate dehydrogenase multienzyme complex (PDC), which catalyses the oxidation of pyruvate to acetyl-CoA within the mitochondrion, is diminished in animal models of diabetes. Studies with purified PDC components have suggested that the kinases responsible for inactivating the decarboxylase catalytic subunits of the complex are most efficient in their regulatory role when they are bound to dihydrolipoyl acetyltransferase (E2) subunits, which form the structural core of the complex. We report that the addition of an exogenous E2 subdomain (inner lipoyl domain) to an intact PDC inhibits ATP-dependent inactivation of the complex. By combining molecular modelling, site-directed mutagenesis and biophysical characterizations, we have also identified two amino acid residues in this subdomain (Ile229 and Phe231) that largely determine the magnitude of this effect.

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