title: Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli.
abstract: Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been overexpressed in E. coli TG2. The high expression of A. vinelandii wild type E2p (20% of cellular protein) and of a mutant enzyme with two lipoyl domains changed the properties of the inner bacterial membrane. This resulted in a solubilization of A. vinelandii E2p after degradation of the outer membrane by lysozyme without any contamination by E. coli pyruvate dehydrogenase complex (PDC) or other high-molecular-weight contaminants. The same effect could be detected for A. vinelandii E2o, an E2 which contains only one lipoyl domain, whereas almost no solubilization of A. vinelandii E2p with one lipoyl domain or of E2p consisting only of the binding and catalytic domain was found. Partial or complete deletion of the alanine- and proline-rich sequence between the binding and the catalytic domain did also decrease the solubilization of the E2p-mutants after lysozyme treatment. Immunocytochemical experiments on E. coli TG2 cells expressing A. vinelandii wild type E2p indicated that the enzyme was present as a soluble protein in the cytoplasm. In contrast, overexpressed A. vinelandii E2p with deletion of all three lipoyl domains and E. coli wild type E2p aggregated intracellularly. The solubilization by lysozyme is therefore ascribed to excluded volume effects leading to changes in the properties of the inner bacterial membrane.
authors.affiliation: Department of Biochemistry, Agricultural University, Wageningen, Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: Schulze
authors.forename: A H
authors.initials: AH
authors.lastname: Westphal
authors.forename: M
authors.initials: M
authors.lastname: Veenhuis
authors.forename: A
authors.initials: A
authors.lastname: de Kok
mesh_terms: D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase