The Pyruvate Dehydrogenase Complex of Escherichia coli K12

Abstract

The nucleotide sequence of the aceF gene, which encodes the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase complex of Escherichia coli Kl2, has been determined using the dideoxy chaintermination method. The aceFgene comprises 1887 base pairs (629 codons excluding the initiation codon AUG); it is preceded by a short intercistronic segment of 14 base pairs containing a good ribosomal binding site, and it is followed closely by a potential rho-independent terminator. The results extend by 1980 base pairs the previously sequenced segment of 3780 base pairs containing the structural gene (ace@ of the pyruvate dehydrogenase component (El) and they confirm that aceE and aceF are the proximal and distal genes of the ace operon.The amino terminus, carboxy-terminal sequence and amino acid composition of the acetyltransferase subunit predicted from the nucleotide sequence are in excellent agreement with previous studies with the purified protein. The predicted molecular weight ( M , = 65959) confirms experimental values derived from sedimentation equilibrium analysis and indicates that the higher values (78 000 -89 000) that have been reported are due to unusual features of the protein that lead to anomalous mobilities during sodium dodecyl sulphate/polyacrylamide gel electrophoresis and in gel filtration. The primary structure fully supports conclusions, based on limited tryptic proteolysis, that the acetyltransferase subunit possesses two heterologous domains : the lipoyl domain and the subunit binding and catalytic domain. The lipoyl domain corresponds to the amino-terminal segment of the protein. It is acidic and contains three remarkably homologous repeating units of approximately 100 amino acids, each possessing a potential lipoyl binding site and a region that is characteristically rich in alanine and proline residues. The subunit binding and catalytic domain occupies most of the residual polypeptide in the carboxy-terminal segment.The pyruvate dehydrogenase complex catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA and CO, via a series of enzyme-bound intermediates [I, 21.It contains multiple copies of three cnzymic components: pyruvate dehydrogenase (E I), dihydrolipoamide acetyltransferase (E2) and lipoamide dehydrogenase (E 3). In Esclzerichiu coli the acetyltransferase component contains 24 identical polypeptide chains forming the structural core of the complex to which the El and E3 subunits are independently bound. The E2 component contains covalently-bound lipoyl cofactors and it participates in the generation of acetyl groups from hydroxyethyl-TPP-El and their transfer to coenzyme A.where TPP is thiamin pyrophosphate. The apparent molecular weight of the E2 subunit has been estimated as 78000 -89000 by sodium dodecyl sulphate/ polyacrylamide gel electrophoresis or gel-filtration in gua- nidine HCI using either purified protein [3 -91 or specificallylabelled products of phage-directed protein synthesis [lo]. However, values of 60000 -65 000 have been obtained b...

The Dihydrolipoamide S-Acetyltransferase Subunit of the Mitochondrial Pyruvate Dehydrogenase Complex from Maize Contains a Single Lipoyl Domain

“…The E2 subunit forms the core to which all other PDC subunits bind (10). In Gram-negative bacteria such as Escherichia coli, the oligomeric core contains eight E2 trimers arranged as a cube with octahedral symmetry (11), while in Gram-positive bacteria (12,13) and eukaryotes (14,15) the core contains 20 E2 trimers arranged as a pentagonal dodecahedron with icosahedral symmetry. Amino acid sequence alignments comparing E2s from divergent organisms revealed that they are multi-domain proteins (see Ref.…”

Nuclear Magnetic Resonance Evidence for the Role of the Flexible Regions of the E1 Component of the Pyruvate Dehydrogenase Complex from Gram-negative Bacteria

“…[3][4][5]. The thiamin diphosphate (ThDP)-dependent E1 component (E1ec, pyruvate dehydrogenase) is an ␣ 2 homodimer and carries out consecutively pyruvate decarboxylation and reductive acetylation of the E2 component (Equation 3).…”

In vivo assembly of yeast mitochondrial alpha-ketoglutarate dehydrogenase complex.

“…Such reconstitutions, carried out mainly with the E. coli complex, have been instrumental in demonstrating the existence of a high-molecular-weight core particle composed of KE2 onto which are attached dimeric units of KE1 and E3 (18,31). Knowledge of the primary structures of the components of KGDC (4,5,28) and the pyruvate dehydrogenase complex (PDC) (15,29,30), gained from the cloned genes, has contributed new insights about the domains involved in catalytic activity and subunit interaction (16,17,36).…”

“…Such reconstitutions, carried out mainly with the E. coli complex, have been instrumental in demonstrating the existence of a high-molecular-weight core particle composed of KE2 onto which are attached dimeric units of KE1 and E3 (18,31). Knowledge of the primary structures of the components of KGDC (4, 5, 28) and the pyruvate dehydrogenase complex (PDC) (15,29,30), gained from the cloned genes, has contributed new insights about the domains involved in catalytic activity and subunit interaction (16,17,36).Despite the large body of information bearing on structure and function relationships in KGDC and PDC, little attention has been paid in the past to the mechanisms ensuring a coordinate expression of their constituent enzymes and the * Corresponding author. manner in which they interact with one another in vivo to form the functional complexes.…”