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Site-directed mutagenesis was performed in the protease-sensitive region, between the lipoyl and catalytic domains and in the catalytic domain, of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. The interaction of the mutated enzymes with the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) was studied by gel filtration experiments, analytical ultracentrifugation and reconstitution of the pyruvate dehydrogenase complex. Upon binding of peripheral components, the 24-subunit core of A. vinelandii wild-type E2p dissociates into tetramers. Four E1p or E3 dimers can bind to a tetramer. Binding is mutually exclusive, resulting in an active complex containing one E3 and three E1p dimers. Large deletions of the protease-sensitive region of E2p resulted in a total loss of the E1p and E3 binding. A small deletion (Δ P361–R362) or the point mutation K367Q in the protease-sensitive region did not influence E3 binding, but affected E1p binding strongly, although with excess E1p almost complete reconstitution was reached. For E2p with the point mutation R416D in the N-terminal region of the catalytic domain only 16% overall activity could be measured in reconstituted complexes. This is due to a very weak E1p/E2p interaction, whereas the E3 binding was not affected. The point mutation R416D did not influence the catalytic activity of E2p, although a function for this residue in the formation of the active site was predicted from amino acid similarities with chloramphenicol acetyltransferase type III from Escherichia coli. Deletion of the complete Ala + Pro-rich sequence between the protease-sensitive region and the catalytic domain did not affect the enzymological properties of E2p, nor the affinity for E1p or E3. A further deletion of 20 N-terminal residues from the catalytic domain destroyed the E2p activity. From gel filtration experiments it was concluded that the quaternary structure was unaffected, as was E3 binding. E1p binding was lost and, in contrast to the wild-type enzyme, no dissociation of the core upon addition of E3 was observed. This mutant enzyme possesses, like E. coli E2p, six E3 binding sites and clearly shows that interaction of E3 or E1p with the E1p sites and dissociation are linked processes. It is concluded that the binding site for E3 is located on the N-terminal part of the protease-sensitive region. In contrast, the binding site for E1p consists of two regions, one located on the protease-sensitive region and one of the catalytic domain. These regions are separated by a flexible sequence of about 20 amino acids. [ABSTRACT FROM AUTHOR]

Partial sequences of the dihydrolipoyl transacetytase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli, containing the catalytic domain, were cloned in pUC plasmids and over-expressed in E. coli TG2. A high expression of a homogeneous protein was only detectable for E2p mutants consisting of the catalytic domain and the alanine-proline-rich sequence between a putative binding region for the peripheral components and the catalytic domain(apa-4). Most of the catalytic domain from A. vinelandii without the apa-4 sequence was degraded intracellularly, probably due to incorrect folding. Fusion proteins of six amino acids from β-galactosidase, the apa-4 region and the catalytic domains of ,4, vinelandii or E. coli E2p could be highly purified. Both catalytic domains were assembled in 24-subtmit structures with a molecular mass of approximately 670 kDa. The expression of catalytic domain from A. vinelandii E2p is more than twice as high as found for wild-type E2p. This can be explained by intracellular degradation of overexpressed wild-type E2p, whereas the catalytic domains are stable against proteolysis/n vivo and in vitro. The interaction of the peripheral components pyruvate dehydrogenase (E1p) and dihydrolipoamide dehydrogenase (E3) with the catalytic domains was studied, using gel filtration on Superose-6 and sedimentation velocity experiments. No binding of either E1p or E3 to the catalytic domain of either organism was detectable. Crystals of the catalytic domain of A. vinelandii E2p could ix: grown to a maximum size of 0.6 × 0.6 times; 0.4 mm. They diffract up to a resolution of 0.28 nm. [ABSTRACT FROM AUTHOR]

The interaction between lipoamide dehydrogenase (E3) and dihydrolipoyl transacetylase (E2p) from the pyruvate dehydrogenase complex was studied during the reconstitution of monomeric E3 apoenzymes from Azotobacter vinelandii and Pseudomonas fluorescens. The dimeric form of E3 is not only essential for catalysis but also for binding to the E2p core, because the apoenzymes as well as a monomeric holoenzyme from P. fluorescens, which can be stabilized as an intermediate at 0°C, do not bind to E2p. Lipoamide dehydrogenase from A. vinelandii contains a C-terminal extension of 15 amino acids with respect to glutathione reductase which is, in contrast to E3, presumably not part of a multienzyme complex. Furthermore, the last 10 amino acid residues of E3 are not visible in the electron density map of the crystal structure and are probably disordered. Therefore, the C-terminal tail of E3 might be an attractive candidate for a binding region. To probe this hypothesis, a set of deletions of this part was prepared by site-directed mutagenesis. Deletion of the last five amino acid residues did not result in significant changes. A further deletion of four amino acid residues resulted in a decrease of lipoamide activity to 5% of wild type, but the binding to E2p was unaffected. Therefore it is concluded that the C-terminus is not directly involved in binding to the E2p core. Deletion of the last 14 amino acids produced an enzyme with a high tendency to dissociate (Kd approximately 2.5 μM). This mutant binds only weakly to E2p. The diaphorase activity was still high. This indicates, together with the decreased Km for NADH, that the structure of the monomer is not appreciably changed by the mutation. Rather the orientation of the monomers with respect to each other is changed. It can be concluded that the binding region of E3 for E2p is constituted from structural parts of both monomers and binding occurs only when dimerization is complete. [ABSTRACT FROM AUTHOR]

The nucleotide sequence of the gene encoding the 2-oxoglutarate dehydrogenase component (Elo) of the 2- oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined. The protein-coding se- quence consists of 2832 bp (944 codons, including the AUG start codon and the UAA stop codon). The predicted molecular mass (105687 Da) is in good agreement with that published for the isolated enzyme. The Elo gene is separated from the gene encoding the E2o component by a 42-bp intergenic region. No Escherichia-coli-like promoter sequences are found in the sequenced 97 bp upstream from the Elo gene. A putative ribosome-binding site is located 10–16 bp upstream from the start codon of the Elo gene. No terminator sequences could be detected downstream from the stop codon. Together with the identical situation for the E2o gene and the presence of terminating sequences downstream of the E3 gene, it can be assumed that all three genes of the 2-oxoglutarate dehydrogenase multienzyme complex are transcribed as a single mRNA transcript under the control of a promoter, located more than 100 bp upstream of the Elo gene, analogous to the pyruvate dehydrogenase complex in E. coli. The similarity with the sucA gene of E. coli is high with 59% identity. [ABSTRACT FROM AUTHOR]

The article offers information on the disease multiple endocrine neoplasia type 2. It mentions that RET-mutation screening is a test available for familial MTC. It also mentions that the clinical specificity of the disease dependents on variable factors such as family history or age. Further it mentions that multiple endocrine neoplasia type 2 is also known as multiple endocrine neoplasia 2A, multiple endocrine neoplasia 2B (MEN 2B), and familial medullary thyroid carcinoma (FMTC).

Medullary thyroid carcinoma (MTC) is a rare calcitonin producing tumor. About 70-75% of patients with MTC have sporadic disease while the others suffer from hereditary MTC. Hereditary MTC is divided into three clinical subtypes: multiple endocrine neoplasia (MEN) type 2A is characterized by MTC, pheochromocytoma and primary hyperparathyroidism. MEN 2B is characterized by aggressive MTC, pheochromocytoma, marfanoid habitus and the presence of distinctive mucosal neuromas on the tongue, lips and subconjunctival areas as well as ganglioneuromatosis of the gastrointestinal tract. The third clinical subtype of inherited MTC, familial MTC, is defined as the presence of MTC in families without evidence of adrenal or parathyroid gland involvement. Hereditary MTC is caused by autosomal dominant gain-of-function mutations in the RET proto-oncogene. The first RET germline mutations were identified in 1993 in patients with MEN 2A and FMTC. Initially a codon 634 (exon 11) mutation was found in approximately 85% of patients with MEN 2A, and germline mutations in FMTC kindreds were more equally distributed throughout the RET proto-onocogene. In about 5% of families in these earlier series, mutations did not reside in exons 10 and 11. We now report a change in the spectrum of mutations detected in the RET proto-oncogene in patients with hereditary MTC from the 'classical' mutation at codon 634 in exon 11 (level 2) to more cases with mutations in the exons 13-15 (level 1) and less aggressive disease. In our series 38.9% of mutations were level 1 mutations, 54.4% level 2, and 5.6% level 3 mutations.