title: Isolation and characterization of lipoylated and unlipoylated domains of the E2p subunit of the pyruvate dehydrogenase complex of Escherichia coli.
abstract: The dihydrolipoamide acetyltransferase subunit (E2p) of the pyruvate dehydrogenase complex of Escherichia coli has three highly conserved and tandemly repeated lipoyl domains, each containing approx. 80 amino acid residues. These domains are covalently modified with lipoyl groups bound in amide linkage to the N6-amino groups of specific lysine residues, and the cofactors perform essential roles in the formation and transfer of acetyl groups by the dehydrogenase (E1p) and acetyltransferase (E2p) subunits. A subgene encoding a hybrid lipoyl domain was previously shown to generate two products when overexpressed, whereas a mutant subgene, in which the lipoyl-lysine codon is replaced by a glutamine codon, expresses only one product. A method has been devised for purifying the three types of independently folded domain from crude extracts of E. coli, based on their pH-(and heat-)stabilities. The domains were characterized by: amino acid and N-terminal sequence analysis, lipoic acid content, acetylation by E1p, tryptic peptide analysis and immunochemical activity. This has shown that the two forms of domain expressed from the parental subgene are lipoylated (L203) and unlipoylated (U203) derivatives of the hybrid lipoyl domain, whereas the mutant subgene produces a single unlipoylatable domain (204) containing the Lys-244----Gln substitution.
authors.affiliation: Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, U.K.
authors.forename: S T
authors.initials: ST
authors.lastname: Ali
authors.forename: J R
authors.initials: JR
authors.lastname: Guest
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000032:analysis* / Q000235:genetics; D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000494:pharmacology; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011768:Pyruvate Dehydrogenase Complex / Q000032:analysis*; D008063:Thioctic Acid / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial; D014357:Trypsin / Q000378:metabolism
chemical_list: D011768:Pyruvate Dehydrogenase Complex; D008063:Thioctic Acid; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D014357:Trypsin; D008239:Lysine; D000645:Ammonium Sulfate