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

Abstract

Limited proteolysis with trypsin has been used to study the domain structure of the dihydrolipoyltransacetylase (E2) component of the pyruvate dehydrogenase complex of Azotobacter vinelundii. Two stable end products were obtained and identified as the N-terminal lipoyl domain and the C-terminal catalytic domain. By performing proteolysis of E2, which was covalently attached via its lipoyl groups to an activated thiol-Sepharose matrix, a separation was obtained between the catalytic domain and the covalently attached lipoyl domain. The latter was removed from the column after reduction of the S-S bond and purified by ultrafiltration. The lipoyl domain is monomeric with a mass of 32.6 kDa. It is an elongated structure with flf0 = 1.62. Circulair dichroic studies indicates little secondary structure. The catalytic domain is polymeric with s20.w = 17 S and mass = 530 kDa. It is a compact structure withflh = 1.24 and shows 40% of the secondary structure of E2. The cubic structure of the native E2 is retained by this fragment as observed by electron microscopy. Ultracentrifugation in 6 M guanidine hydrochloride in the presence of 2 mM dithiothreitol yields a mass of 15.8 kDa. An N-terminal sequence of 36 amino acids is homologous with residues 370-406 of Escherichiu coli E2. The catalytic domain possesses the catalytic site, but in contrast to the E. coli subunit binding domain the pyruvate dehydrogenase (El) and lipoamide dehydrogenase (E3) binding sites are lost during proteolysis. From comparison with the E. coli E2 sequence a model is presented in which the several functions, such as lipoyl domain, the E3 binding site, the catalytic site, the Ez/E2 interaction sites, and the El binding site, are indicated.The pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate resulting in the production of acetyl-coenzyme A and NADH. In gram-negative bacteria three enzymes take part in this reaction: pyruvate dehydrogenase (El), lipoylacetyltransferase (E2) and lipoamide dehydrogenase (E3). El and E3 are non-covalently bound as dimers to a core of E2 [l]. The structure of PDC from Azotobucter vinelundii is based on a tetrameric core of E2, to which three El dimers and one E3 dimer are bound [2]. This quaternary structure differs considerably from that of the Escherichiu coli PDC which is based on a core of 24 E2 chains arranged in a cubic structure with 432 symmetry [3]. To this core twelve El dimers and six E3 dimers are bound in the catalytically optimum stoichiometry [4]. Upon removal of the peripheral components, the A . vinelundii E2 associates to a multimer. Although its appearance in the electron microscope is similar to that of E. coli E2, hydrodynamic studies indicate differences in quaternary structure [5, 61. Experiments with hybrid E. coli/A. vinelandii PDC complexes Correspondence f a A.

The dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii

“…About 3% of the known genes possesses the GUG start codon [27]. The molecular mass of the protein deduced from the open reading frame corresponds to that determined by sedimentation equilibrium analysis on the purified enzyme in 6 M guanidine hydrochloride [3]. One other open reading frame was identified preceding the gene encoding E2.…”

“…2 AvIip3 PQEVKVPDIGSAGKARVIEVLVKAGDQVQAEQSLIVLESDKASMEIPSPAAGWESVAVQ ***** * *** *** ** ** * **** ***** *** *** ** * formylmethionine residue is removed post-translationally, so that the serine residue corresponds to the first residue of the N-terminal amino acid sequence that has been determined by automated Edman degradation of the purified protein as isolated from A . vinelandii [3]. An internal sequence of 36 residues, obtained by Edman degradation of the N-terminus of the catalytic domain [3], corresponds exactly with the sequence 381 -416.…”

“…vinelandii [3]. An internal sequence of 36 residues, obtained by Edman degradation of the N-terminus of the catalytic domain [3], corresponds exactly with the sequence 381 -416. Only the last residue proved to be an arginyl residue instead of the reported methionyl residue.…”

“…Antisera were collected as described in [3]. Because of cross-reactivity with E. coli E2, antisera were saturated with E. coli TG2 cell-free extract before use.…”

Lipoamide dehydrogenase from Azotobacter vinelandii

“…Because part of the internal protein sequence of A . vinelandii acetyltransferase (homologous with amino acid residues 370 -406 of the E. coli enzyme) has been determined by Edman degradation [29], the first reading frame could be excluded as coding for acetyltransferase. This was further confirmed by the cloning and sequence analysis of the acetyltransferasqgene of A .…”