scholarly journals Subgenes expressing single lipoyl domains of the pyruvate dehydrogenase complex of Escherichia coli

1987 ◽  
Vol 245 (3) ◽  
pp. 869-874 ◽  
Author(s):  
J S Miles ◽  
J R Guest
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Transcriptional Control ◽  
Expression Vector ◽  
Pyruvate Dehydrogenase Complex ◽  
The Other ◽  
Temperature Sensitive ◽  
Lambda Repressor ◽  
Site Specific ◽  
Dehydrogenase Complex

Subgenes encoding the lipoyl domains from the acetyltransferase components of two types of pyruvate dehydrogenase complex of Escherichia coli were made by site-specific oligonucleotide-directed nonsense mutagenesis of the corresponding aceF genes. One of the domains is capable of binding lipoic acid whereas the other is not. The subgenes were cloned into an expression vector under the transcriptional control of the lambda PL and lambda PR promoters and a temperature-sensitive lambda repressor. Under non-permissive conditions expression of the lipoyl domains was not detected, but 6 h after thermo-induction the domains were amplified by at least 35-50-fold relative to the normal amounts of each type of covalently bound domain.

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.

On The Unique Structural Organization of the Saccharomyces Cerevisiae Pyruvate Dehydrogenase Complex

1998 ◽  
Vol 4 (S2) ◽  
pp. 954-955
Author(s):  
James K. Stoops ◽  
Z. Hong Zhou ◽  
John P. Schroeter ◽  
Steven J. Kolodziej ◽  
R. Holland Cheng ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Pyruvate Dehydrogenase ◽  
Structural Organization ◽  
Pyruvate Dehydrogenase Complex ◽  
The Other ◽  
Oxidative Decarboxylation ◽  
Multienzyme Complex ◽  
The Core ◽  
Dehydrogenase Complex

Dihydrohpoamide acetyl transferase (E2), a catalytic and structural component of a multienzyme complex that catalyzes the oxidative decarboxylation of pyruvate, forms the central core to which the other components are bound. We have utilized protein engineering and 3-D electron microscopy to study the structural organization of the largest multienzyme complex known (Mr ∼ 107). The structures of the truncated 60-mer core (tE2) and complexes of the tE2 associated with a binding protein (BP), and the BP associated with its dihydrohpoamide dehydrogenase (BP'E3) and the intact E2 associated with BP and the pyruvate dehydrogenase (E1) were determined (Figs. 1 and 2). The tE2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges.Previous studies have given rise to the generally accepted belief that BP and BP'E3 components are bound on the outside of the E2 scaffold and that E1 is similarly bound to the core in variable positions by flexible tethers.

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