scholarly journals Domain structure and1H-n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Bacillus stearothermophilus

1984 ◽  
Vol 217 (1) ◽  
pp. 219-227 ◽  
Author(s):  
L C Packman ◽  
R N Perham ◽  
G C K Roberts
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Sites ◽  
Bacillus Stearothermophilus ◽  
Inner Core ◽  
Polypeptide Chain ◽  
Pyruvate Dehydrogenase Complex ◽  
Terminal Region ◽  
Enzyme Complex ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

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.

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.

The pyruvate dehydrogenase multi-enzyme complex of Escherichia coli : genetic reconstruction and functional analysis of the lipoyl domains

1986 ◽  
Vol 317 (1540) ◽  
pp. 391-404 ◽  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Inner Core ◽  
Polypeptide Chain ◽  
Pyruvate Dehydrogenase Complex ◽  
Site Directed Mutagenesis ◽  
Enzyme Complex ◽  
Site Selective

The dihydrolipoamide acetyltransferase (E2p) component of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous lipoyl domains ( ca . 100 residues) that are tandemly repeated to form the N-terminal half of the polypeptide chain. These lipoyl domains are linked to a much larger ( ca . 300 residues) subunit-binding domain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. Selective in vitro deletions in the E2p gene ( aceF )have allowed the creation of truncated E2p chains in which one or more of the lipoyl domains has been excised. Site-directed mutagenesis has been used to change individual residues. The effects of these deletions and mutations on the assembly, catalytic activity and active-site coupling in the complex are assessed.

scholarly journals Purificaton of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and resolution of its four component polypeptides

1980 ◽  
Vol 189 (1) ◽  
pp. 161-172 ◽  
Author(s):  
C E Henderson ◽  
R N Perham
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Pyruvate Dehydrogenase Complex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Icosahedral Symmetry ◽  
Enzyme Complex ◽  
Molecular Masses ◽  
Dehydrogenase Complex

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.

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.

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