Expression in Escherichia coli of a sub-gene encoding the lipoyl and peripheral subunit-binding domains of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.
A sub-gene encoding the N-terminal 170 residues of the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. The expressed polypeptide consists of the lipoyl domain, inter-domain linker and peripheral subunit-binding domain; these were found to have folded into their native functional conformations as judged by reductive acetylation of the lipoyl domain, limited proteolysis of the linker region and ability to bind the dihydrolipoamide dehydrogenase dimer. The di-domain was largely (80%) unlipoylated; a small proportion (4%) was correctly modified with lipoic acid and the remainder (16%) was aberrantly modified with octanoic acid. A polyclonal antiserum was raised that recognized both the di-domain and the individual component domains. The 400 MHz 1H-n.m.r. spectrum of the di-domain showed resonances corresponding to those seen in spectra of the lipoyl domain, plus others characteristic of amino acid residues in the flexible linker region. Further, as yet unidentified, resonances are likely to be derived from the peripheral subunit-binding domain. The existence and independent folding of the peripheral subunit-binding domain is thus confirmed and its purification in large-scale amounts for detailed structural analysis is now possible.
Hipps
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Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, U.K.
Perham
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D000107:Acetylation; D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002845:Chromatography; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics*; D015151:Immunoblotting; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics*
D046911:Macromolecular Substances; D011768:Pyruvate Dehydrogenase Complex; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase