The gene encoding dihydrolipoyl transacetylase from Azotobacter vinelandii. Expression in Escherichia coli and activation and isolation of the protein.
The gene encoding the dihydrolipoyl transacetylase (E2) component from Azotobacter vinelandii has been cloned in Escherichia coli. High expression of the gene was found when the cells were grown for more than 14 h. The E2 produced was partially active, varying 10 and 90% in different experiments. By limited proteolysis of the protein it was shown that the catalytic domain was incorrectly folded, caused by formation of intermolecular or intramolecular S-S bridges. The enzyme was fully activated after unfolding in 2.5 M guanidine hydrochloride containing 2 mM dithiothreitol, followed by refolding by dialysis. Active E2 was isolated in a simple three-step procedure. It possessed a specific activity in the same order as that found after isolation of E2 from purified pyruvate dehydrogenase complex from A. vinelandii. Active E2 comprises about 7% of the total soluble cellular protein in the E. coli clone. By genetic manipulation, deletion mutants of E2 were created, one encoding the lipoyl domain and the N-terminal half of the pyruvate-dehydrogenase (E1)- and lipoamide-dehydrogenase (E3)-binding domain, the other encoding the catalytic domain and the C-terminal half of the E1- and E3-binding domain. In E. coli expression of both mutants was observed.
Hanemaaijer
R
R
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Westphal
A H
AH
Berg
A
A
Van Dongen
W
W
de Kok
A
A
Veeger
C
C
D000123:Acetyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001395:Azotobacter / Q000201:enzymology / Q000235:genetics*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids; D011092:Polyethylene Glycols; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase