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

Abstract

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...

Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli

“…On the other hand, limited proteolysis of E. coli dihydrolipoyltranssuccinylase (E2o) resulted in loss of the E3-binding, whereas Elo was still bound to the catalytic domain [14]. Upon binding of Elp and E3, the E2p from A. cinelandii behaves in a unique way, because its core structure of 24 subunits dissociates into smaller structures [15,16]. E2p from E. coli does not dissociate upon binding of Elp and/or E3 [9].…”

Atomic Structure of the Cubic Core of the Pyruvate Dehydrogenase Multienzyme Eomplex

“…The PDC from Gram-negative Azotobacter vinelandii is well characterized (18). The isolated E2p core consists of 24 subunits, The region shown comprises the residues in the active center.…”

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

“…vinelandii E2p is a 24-subunit assembly, binding of E l p or excess E3 results in dissociation. From previous investigations it was concluded that the dissociated complex is based on a tetrameric core [18,19], but the catalytic domain structure shows that intratrimer interactions are very extensive, while intertrimer interactions are weak. Therefore, the A .…”