Expression of genes encoding the E2 and E3 components of the <i>Bacillus stearothermophilus</i> pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly <i>in vitro</i>

Lessard, Ivan A. D.; Domingo, Gonzalo J.; Borges, Arnaldo Chaer; Perham, Richard N.

doi:10.1046/j.1432-1327.1998.2580491.x

Cited by 49 publications

(52 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Sample Preparation-E2E3 complexes containing 60 copies of fulllength E2 dihydrolipoyl acetyltransferase and 60 copies of the E3 dihydrolipoyl dehydrogenase homodimer were prepared as described (26) by individually expressing the full-length recombinant genes in Escherichia coli and purifying the protein products, which were then reconstituted together in a 1:1 stoichiometric ratio and isolated from unbound E3 by size exclusion chromatography. Aliquots of the complex at ϳ2 mg/ml were frozen in liquid nitrogen and stored at Ϫ80°C until use.…”

Section: Methodsmentioning

confidence: 99%

Molecular Structure of a 9-MDa Icosahedral Pyruvate Dehydrogenase Subcomplex Containing the E2 and E3 Enzymes Using Cryoelectron Microscopy

Milne

Borgnia

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (␣ 2 ␤ 2 ) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. J., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587-5598). An annular gap of ϳ90 Å separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of ϳ75 Å between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain. The pyruvate dehydrogenase (PDH)2 multienzyme complexes play a central role in cellular metabolism, catalyzing the oxidative decarboxylation of pyruvate to acetyl CoA, to link glycolysis and the tricarboxylic acid cycle. Of key medical importance in several metabolic disorders (1, 2), the chemistry of this multicomponent enzyme assembly has been extensively studied (3-5). Detailed structures of individual enzymes or their subdomains have been elucidated using x-ray crystallographic and NMR techniques (Ref. 5 and references therein and Refs. 6 and 7), whereas cryoelectron microscopic analyses have begun to illuminate the overall molecular architecture of PDH complexes from eukaryotes (8 -11) and bacteria (12-14). These systems are ideal paradigms for the structural study of highly dynamic macromolecular machines because they mediate efficient transport of reaction intermediates between the active sites of component enzymes that can be spatially separated by as much as 100 Å (10, 14).PDH complexes based on icosahedral symmetry are among the largest of cellular machines and are found in the mitochondria of eukaryotes and in Gram-positive bacteria such as Bacillus stearo...

show abstract

Section: Methodsmentioning

confidence: 99%

Molecular Structure of a 9-MDa Icosahedral Pyruvate Dehydrogenase Subcomplex Containing the E2 and E3 Enzymes Using Cryoelectron Microscopy

Milne

Borgnia

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…A reaction mixture containing 1.2 mM ATP, 1.2 mM MgCl 2 , 0.5 mM sodium lipoate (Sigma), 0.1 mg/ ml LplA and 10-300 nmol of lipoyl domain in 20 mM Tris-HCl (pH 7.5), was incubated at 25 C for three hours (Lessard et al, 1998). The products were separated by anion exchange chromatography using a Resource-Q TM column (Pharmacia) developed with an ammonium bicarbonate gradient, essentially as described by Dardel et al (1990).…”

Section: Lipoylation Of the E Coli Lipoyl Domains In Vitromentioning

confidence: 99%

Structural determinants of post-translational modification and catalytic specificity for the lipoyl domains of the pyruvate dehydrogenase multienzyme complex of Escherichia coli

Jones

Horne

Reche

et al. 2000

Journal of Molecular Biology

View full text Add to dashboard Cite

The lipoyl domains of the dihydrolipoyl acyltransferase (E2p, E2o) components of the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes are speci®cally recognised by their cognate 2-oxo acid decarboxylase (E1p, E1o). A prominent surface loop links the ®rst and second bstrands in all lipoyl domains, close in space to the lipoyl-lysine b-turn. This loop was subjected to various modi®cations by directed mutagenesis of a sub-gene encoding a lipoyl domain of Escherichia coli E2p. Deletion of the loop (four residues) rendered the domain incapable of reductive acetylation by E. coli E1p in the presence of pyruvate, but insertion of a new loop (six residues) corresponding to that in the E2o lipoyl domain partly restored this ability, albeit with a much lower rate. However, the modi®ed domain remained unable to undergo reductive succinylation by E1o in the presence of 2-oxoglutarate. Additional exchange of the two residues on the C-terminal side of the loop (V14A, E15T) had no effect. Insertion of a different four-residue loop also restored a limited ability to undergo reductive acetylation, but still signi®cantly less than that of the wild-type domain. Exchanging the residue on the N-terminal side of the lipoyl-lysine b-turn in the E2p and E2o domains (G39T), both singly and in conjunction with the loop exchange, had no effect on the ability of the E2p domain to be reductively acetylated but did confer a slight increase in susceptibility to reductive succinylation. All mutant E2p domains, apart from that with the loop deletion (LD), were readily lipoylated in vitro by E. coli lipoate protein ligase A; the E2p LD mutant could be lipoylated only at a signi®cantly lower rate. Likewise, this domain exhibited 1D and 2D NMR spectra characteristic of a partially folded protein, whereas the spectra of mutants with modi®ed loops were similar to those of the wild-type domain. The surface loop is evidently important to the structural integrity of the domain and may help to stabilize the thioester bond linking the acyl group to the reduced lipoyl-lysine swinging arm as part of the catalytic mechanism. Recognition of the lipoyl domain by its partner E1 appears to be a complex process and not attributable to any single determinant on the domain.

show abstract

“…Therefore, we have investigated both the feasibility of introducing non-native, functional modifications, as well as the impact of these alterations on the structural and thermal stabilities of our engineered scaffold. While the catalytic mechanisms and biology of the pyruvate dehydrogenase complex from Bacillus stearothermophilus has been well-studied (Allen and Perham, 1997;Domingo et al, 2001Domingo et al, , 2003Izard et al, 1999;Jung et al, 2002;Lessard et al, 1998;Milne et al, 2002Milne et al, , 2006Perham, 1991), the potential application of its E2 structural scaffold as a molecular carrier has not been investigated.…”

Section: Introductionmentioning

confidence: 99%

Thermostability and molecular encapsulation within an engineered caged protein scaffold

Dalmau

Lim

Chen

et al. 2008

Biotech & Bioengineering

213

View full text Add to dashboard Cite

Self-assembling biological complexes such as viral capsids have been manipulated to function in innovative nanotechnology applications. The E2 component of pyruvate dehydrogenase from Bacillus stearothermophilus forms a dodecahedral complex and potentially provides another platform for these purposes. In this investigation, we show that this protein assembly exhibits unusual stability and can be modified to encapsulate model drug molecules. To distill the E2 protein down to its structural scaffold core, we synthesized a truncated gene optimized for expression in Escherichia coli. The correct assembly and dodecahedral structure of the resulting scaffold was confirmed with dynamic light scattering and transmission electron microscopy. Using circular dichroism and differential scanning calorimetry, we found the thermostability of the complex to be unusually high, with an onset temperature of unfolding at 81.1 +/- 0.9 degrees C and an apparent midpoint unfolding temperature of 91.4 +/- 1.4 degrees C. To evaluate the potential of this scaffold for encapsulation of guest molecules, we made variants at residues 381 and 239 which altered the physicochemical properties of the hollow internal cavity. These mutants, yielding 60 and 120 mutations within this cavity, assembled into the correct architecture and exhibited high thermostability that was comparable to the wild-type scaffold. To show the applicability of this scaffold, two different fluorescent dye molecules were covalently coupled to the cysteine mutant at site 381. We demonstrate that these mutations can introduce non-native functionality and enable molecular encapsulation within the cavity while still retaining the dodecahedral structure. The unusually robust nature of this scaffold and its amenability to internal changes reveal its potential for nanoscale applications.

show abstract

Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro

Cited by 49 publications

References 37 publications

Molecular Structure of a 9-MDa Icosahedral Pyruvate Dehydrogenase Subcomplex Containing the E2 and E3 Enzymes Using Cryoelectron Microscopy

Molecular Structure of a 9-MDa Icosahedral Pyruvate Dehydrogenase Subcomplex Containing the E2 and E3 Enzymes Using Cryoelectron Microscopy

Structural determinants of post-translational modification and catalytic specificity for the lipoyl domains of the pyruvate dehydrogenase multienzyme complex of Escherichia coli

Thermostability and molecular encapsulation within an engineered caged protein scaffold

Contact Info

Product

Resources

About