scholarly journals Interactions of the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

2003 ◽  
Vol 270 (22) ◽  
pp. 4488-4496 ◽  
Author(s):  
Hyo-Il Jung ◽  
Alan Cooper ◽  
Richard N. Perham
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Binding Domain ◽  
Multienzyme Complex ◽  
Dihydrolipoyl Acetyltransferase

scholarly journals Interaction of component enzymes with the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: stoichiometry and specificity in self-assembly

1995 ◽  
Vol 306 (3) ◽  
pp. 727-733 ◽  
Author(s):  
I A D Lessard ◽  
R N Perham
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Self Assembly ◽  
Bacillus Stearothermophilus ◽  
Pyruvate Decarboxylase ◽  
Binding Domain ◽  
Alpha Subunit ◽  
Multienzyme Complex ◽  
Beta 2 ◽  
Dihydrolipoyl Acetyltransferase

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.

scholarly journals 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

1994 ◽  
Vol 297 (1) ◽  
pp. 137-143 ◽  
Author(s):  
D S Hipps ◽  
L C Packman ◽  
M D Allen ◽  
C Fuller ◽  
K Sakaguchi ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Pyruvate Dehydrogenase Complex ◽  
Tight Binding ◽  
Limited Proteolysis ◽  
Binding Domain ◽  
Dihydrolipoyl Acetyltransferase ◽  
Dihydrolipoyl Dehydrogenase ◽  
Lipoyl Domain

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was released by limited proteolysis from a di-domain (lipoyl domain plus binding domain) encoded by a subgene over-expressed in Escherichia coli. The domain was characterized by N-terminal sequence analysis, electrospray m.s. and c.d. spectroscopy. It was found to be identical in all respects to a chemically synthesized peptide of the same sequence. The association of the di-domain and binding domain (both natural and synthetic) with dihydrolipoyl dehydrogenase was analysed in detail and a tight binding was demonstrated. As judged by several different techniques, it was found that only one peripheral subunit-binding domain is bound to one dimer of dihydrolipoyl dehydrogenase, implying that the association is highly anti-cooperative.

scholarly journals Expression in Escherichia coli of a sub-gene encoding the lipoyl and peripheral subunit-binding domains of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus

1992 ◽  
Vol 283 (3) ◽  
pp. 665-671 ◽  
Author(s):  
D S Hipps ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Large Scale ◽  
Bacillus Stearothermophilus ◽  
Pyruvate Dehydrogenase Complex ◽  
Limited Proteolysis ◽  
Binding Domain ◽  
Gene Encoding ◽  
Linker Region ◽  
Lipoyl Domain

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.

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