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

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj2830665 ◽

1992 ◽

Vol 283 (3) ◽

pp. 665-671 ◽

Cited By ~ 12

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.

Download Full-text

Amino acid sequence analysis of the lipoyl and peripheral subunit-binding domains in the lipoate acetyltransferase component of the pyruvate dehydrogenase complex from Bacillus stearothermophilus

Biochemical Journal ◽

10.1042/bj2520079 ◽

1988 ◽

Vol 252 (1) ◽

pp. 79-86 ◽

Cited By ~ 46

Author(s):

L C Packman ◽

A Borges ◽

R N Perham

Keyword(s):

Amino Acid ◽

Sequence Analysis ◽

Pyruvate Dehydrogenase ◽

Bacillus Stearothermophilus ◽

Pyruvate Dehydrogenase Complex ◽

Limited Proteolysis ◽

Binding Domain ◽

British Library ◽

Amino Acid Residues ◽

Lipoyl Domain

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj3060727 ◽

1995 ◽

Vol 306 (3) ◽

pp. 727-733 ◽

Cited By ~ 27

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj3340703 ◽

1998 ◽

Vol 334 (3) ◽

pp. 703-711 ◽

Cited By ~ 17

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.

Download Full-text

Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries

Biochemical Journal ◽

10.1042/bj1730659 ◽

1978 ◽

Vol 173 (2) ◽

pp. 659-668 ◽

Cited By ~ 67

Author(s):

Peter H. Sugden ◽

Philip J. Randle

Keyword(s):

Pyruvate Dehydrogenase ◽

Gel Electrophoresis ◽

Pyruvate Dehydrogenase Complex ◽

Sodium Dodecyl ◽

Molar Ratio ◽

Α Subunit ◽

Molar Ratios ◽

Dihydrolipoyl Acetyltransferase ◽

Dihydrolipoyl Dehydrogenase ◽

Α Subunits

1. The molecular weights of the subunits of purified pig heart pyruvate dehydrogenase complex were determined by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis and were: pyruvate decarboxylase, α-subunit 40600, β-subunit 35100; dihydrolipoyl acetyltransferase 76100; dihydrolipoyl dehydrogenase 58200. 2. Inactivation of the pyruvate dehydrogenase complex by its integral kinase corresponded to the incorporation of 0.46nmol of P/unit of complex activity inactivated. 3. Further incorporation of phosphate into the complex occurred to a limit of 1.27nmol of P/unit of complex inactivated (approx. 3 times that required for inactivation). 4. Phosphate was incorporated only into the α-subunit of the decarboxylase. 5. The molar ratio of phosphate to α-subunits of the decarboxylase was estimated by radioamidination of amino groups of pyruvate dehydrogenase [32P]phosphate complex by using methyl [1-14C]acetimidate, followed by separation of α-subunits by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis. Inactivation of the complex (0.46nmol of P/unit of complex inactivated) corresponded to a molar ratio of one phosphate group per two α-chains (i.e. one phosphate group/α2β2 tetramer). Complete phosphorylation corresponded to three phosphate groups per α2β2 tetramer. 6. Subunit molar ratios in the complex were also estimated by the radioamidination technique. Results corresponded most closely to molar ratios of 4 α-subunits:4 β-subunits:2 dihydrolipoyl acetyltransferase subunits:1 dihydrolipoyl dehydrogenase subunit.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj2170219 ◽

1984 ◽

Vol 217 (1) ◽

pp. 219-227 ◽

Cited By ~ 43

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.

Download Full-text