The dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii

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After limited proteolysis of the dihydrolipoyl transacetylase component (E,) of Azotobucter vinelandii pyruvate dehydrogenase complex (PDC), a C-terminal domain was obtained which retained the transacetylase active site and the quaternary structure of E2 but had lost the lipoyl-containing N-terminal part of the chain and the binding sites for the peripheral components, pyruvate dehydrogenase and lipoamide dehydrogenase. The C-terminus of this domain was determined by treatment with carboxypeptidase Y and shown to be identical with the C-terminus of E2. Together with the previously determined N-terminus and the known amino acid sequence of E2, a molecular mass of 27.5 kDa was calculated. From the molecular mass of the native catalytic domain, 530 kDa, and the symmetry of the cubic structures observed on electron micrographs, a 24-meric structure is concluded instead of the 32-meric structure proposed previously.From the effect of guanidine hydrochloride on the light-scattering of intact E, it was concluded that dissociation occurs in a two-step reaction resulting in particles with an average mass 1/6 that of thc original mass before the N + D transition takes place. Cross-linking experiments with the catalytic domain indicated that the multimeric E, is built from tetramers and that the tetramers are arranged as a dimer of dimers. A model for the quaternary structure of E2 is given, in which it is assumed that the tetrameric E2 core of PDC is formed from each of the six morphological subunits located at the lateral face of the cube. Binding of peripheral components to a site that interferes with the cubic assembly causes dissociation, resulting in the unique small PDC of A . vinelandii. Azotobacter vinelandii is the smallest complex known [6]. It is based on a tetrameric E2 core [7, 81. The sedimentation coefficient of this complex is 37-19s [7]; that of the complex from E. coli is 53-60s [3, 9, lo]. However, a 37-20s form of E. coli PDC has also been observed [5, 91. This small form is enzymatically active and is present in small amounts in E. coli PDC preparations. It is indicated as having a trimeric core, being the morphological subunit of both the cubic and the icosahedral complexes [ll, 321. On the other hand, upon removal of the peripheral components of the A . vinelandii tetrameric core, E, associates to a multimcr. In electron micrographs a similar cubic appearance as the E. coli E2 core is observed [l, 131. On basis of its molecular mass of about 2 MDa [8, 131 and its association/dissociation behaviour [13] it was thought to be composed of tetrameric instead of trimeric morphological subunits, arranged at the vertices of the E2 cube. This would result in an E2 core composed of 32 subunits The E2 components of PDCs from different organisms are exquisitely sensitive to proteolytic cleavage under nondenaturing conditions [14-171. After proteolysis, a domain comprising the lipoyl moieties and a structural domain are usually obtained. In the latter, the quaternary structure of intact E2 and the transacetyl...

Section: C-terminus Determinationsupporting

confidence: 56%

Section: Protein Purificationmentioning

confidence: 99%

The quaternary structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii

Hanemaaijer¹,

Westphal²,

Heiden³

et al. 1989

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“…The initiating formylmethionine residue must be processed post-translationally from the E2 chain, since the N-terminal sequence of the native protein begins with the succeeding alanine residue [7]. The structural gene for the E2 component of the Azotobucter vinelundii PDH complex also has GTG as initiation codon [18]. ThepdhCgene is composed of 54.6% G/C, with 60.6% of the codons ending in G or C. A putative ribosome-binding site for the pdhC gene, complementary to the 3' end of the 16s rRNA from B.…”

Section: Resultsmentioning

confidence: 99%

Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

Borges

Hawkins

Packman

et al. 1990

A 2641-bp EcoRI fragment of DNA that encodes the C-terminal part of the dihydrolipoyl acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. A 705-bp truncated open reading frame was located at the 5' end of the insert which, together with the 588-bp truncated open reading frame at the 3' end of another EcoRI fragment of B. stearothermophilus DNA previously cloned and sequenced [Hawkins, C. F., Borges, A. & Perham, R. N. (1990) Eur. J. Biochem. 191,, was identified as the gene, pdhC, encoding the E2 polypeptide chain. Direct sequence analysis of the purified E2 chain confirmed that the two EcoRI fragments are adjoining in the B. stearothermophilus genome. The E3 gene, pdhD, begins just 4 bp downstream from the stop codon of the pdhC gene. The amino acid sequences deduced from the pdhC and pdhD genes correspond to proteins of 427 amino acids (E2, M , 46265) and 469 amino acids (E3, M , 49193), respectively. Both genes are preceded by potential ribosome-binding sites and the E3 gene is followed by a stemloop structure characteristic of rho-independent transcription terminators. The B. stearothermophilus E2 and E3 chains exhibit substantial sequence similarity with the corresponding subunits of other 2-0x0-acid dehydrogenase multienzyme complexes. The cloning and sequence analysis described here complete the description of the gene cluster (pdhA, B, C and D ) which encodes the B. stearothermophilus pyruvate dehydrogenase multienzyme complex.The oxidative decarboxylation of 2-0x0-acids is accomplished in most organisms by the 2-0x0-acid dehydrogenase multienzyme complexes, producing the corresponding acyl-CoA and NADH. For the pyruvate dehydrogenase (PDH) complex, the constituent enzymes that function successively in the mechanism are pyruvate dehydrogenase (El), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3) (for recent reviews see [I, 21).The PDH complex from the thermophilic, Gram-positive bacterium Bacillus stearothermophilus is assembled round a core of the E2 component of 60 copies of the E2 polypeptide chain organized with icosahedral symmetry [3]. Multiple cop- ies of the El (comprising two polypeptide subunits, Elcl and Elp) and E3 components are attached to this core tightly but non-covalently. The M, of the individual components as well as the morphology of the E2 core [3, 41 closely resemble those of the PDH complexes of eukaryotes. Its thermostability makes the B. stearothermophilus PDH complex a good system for studying the structure of a 2-0x0-acid dehydrogenase complex based on icosahedral symmetry, which differs from the PDH and 2-oxoglutarate dehydrogenase complexes of Escherichia coli in that the latter have octahedral E2 cores [5, 61. The sequence of the first 21 1 amino acid residues from the N-terminus of the B. stearothermophilus E2 chain has been determined [7], showing that this ace...

“…vinelandii and E. coli E2p, the plasmids pRA282 ( A . vinelandii wild-type E2p) [3] and PAW10 (E. coli wild-type E2p) [6] were used as starting material (Fig. 1 B).…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

“…In the plasmid pRA282 the SmaI site in the polylinker was destroyed and a new SmaI site was introduced by site-directed mutagenesis at position 1598 of the original DNA sequence which encodes the C-terminus of the binding domain [3]. The resulting plasmid was named pER27 [5].…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

Reconstitution of pyruvate dehydrogenase multienzyme complexes based on chimeric core structures from Azotobacter vinelandii and Escherichia coli

Schulze

Westphal

Veeger

et al. 1992

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Two unique restriction sites were introduced by site-directed mutagenesis at identical positions in the DNA encoding the dihydrolipoyltransacetylase (E2p) components of the pyruvate dehydrogenase complex from Azotobacter vinelandii and from Escherichiu coli. In this manner each DNA chain could be cut into three parts, coding for the lipoyl domain, which consists of three lipoyl subdomains, the binding domain and the core-forming catalytic domain, respectively. Chimeric E2p components were constructed by exchanging the three domains between E2p from A . vinelundii and E. coli on gene level. The six chimeric E2p proteins were expressed and purified from E. coli TG2. All chimeras were catalytically active, 24-subunit E2p proteins. Interactions of the peripheral components E l p and E3 with the wild-type enzymes from A . vinelundii and E. coli and with the chimeric proteins were studied by gel-filtration experiments, analytical ultracentrifugation and reconstitution of the overall activity of the complex.A. vinelundii E3 interacts only with those chimeras that contain the A . vinelandii binding domain, whereas E. coli E3 interacts with all chimeras. Exchange of the lipoyl or catalytic domain did not influence the binding properties of E3. Recognition of E l p depends on the origin of both the binding domain and the catalytic domain. E. coli E l p interacts strongly with those chimeras in which both the binding domain and the catalytic domain were derived from E. coli E2p and weakly with chimeras that contained either the binding domain or the catalytic domain from E. coli E2p. No binding of E. coli E l p was observed when both domains were of A . vinelandii origin. A . vinelundii E l p recognizes E2p from A.vinelundii and E. coli, but strong interaction required that the binding and catalytic domain were of the same origin. Exchange of lipoyl domains had no effect on the binding properties of the E l p component. These observations confirm previous conclusions, based on site-directed mutagenesis of A . vinelundii E2p [Schulze, E., Westphal, A. H., Boumans, H. and de Kok, A. (1991) Eur. J . Biochem. 202,, that the binding site for E l p consists of amino acid residues derived from both the binding and the catalytic domain and extend these conclusions to E. coli E2p.Dissociation of the 24 subunit E2p core was only detected when the chimeric E2p proteins contained the catalytic domain from A . vinelundii E2p. Dissociation depends on the binding of peripheral components to the Elp-binding sites, pointing to differences in the inter-trimer contacts between the E2p proteins from both species.Reconstitution of PDC activity depends on saturation by peripheral components under the conditions used and on recognition of the lipoyl domain by the respective active sites. All reconstituted complexes, based on chimeric E2p proteins, regain PDC activity ranging between 16-96% of the wild-type activity. It is concluded that E l p is highly specific with respect to recognition of the lipoyl domain, in contrast to E2p and E3.The pyruv...