Genetic reconstruction and functional analysis of the repeating lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.
The dihydrolipoamide acetyltransferase component (E2p) of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous sequences of about 100 residues that are tandemly repeated to form the N-terminal half of the polypeptide chain. All three sequences include a lysine residue that is a site for lipoylation and they appear to form independently folded functional domains. These lipoyl domains are in turn linked to a much larger (about 300 residues) subunit-binding domain of the E2p chain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. In order to investigate whether individual lipoyl domains play different parts in the enzymic mechanism, selective deletions were made in vitro in the dihydrolipoamide acetyltransferase gene (aceF) so as to excise one or two of the repeating sequences. This was facilitated by the high degree of homology in these sequences, which allowed the creation of hybrid lipoyl domains that closely resemble the originals. Pyruvate dehydrogenase complexes incorporating these genetically reconstructed E2p components were purified and their structures were confirmed. It was found that the overall catalytic activity, the system of active site coupling, and the ability to complement pyruvate dehydrogenase complex mutants, were not significantly affected by the loss of one or even two lipoyl domains per E2p chain. No special role can be attached thus far to individual lipoyl domains. On the other hand, certain genetic deletions affecting the acetyltransferase domain caused inactivation of the complex, highlighting particularly sensitive areas of that part of the E2p chain.
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004926:Escherichia coli / Q000201:enzymology*; D005798:Genes, Bacterial; D005818:Genetic Engineering; D046911:Macromolecular Substances; D009154:Mutation; D010957:Plasmids; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics* / Q000378:metabolism
D046911:Macromolecular Substances; D011768:Pyruvate Dehydrogenase Complex; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase