PMID:12651118
| Citation |
Wei, W, Li, H, Nemeria, N and Jordan, F (2003) Expression and purification of the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase subunits of the Escherichia coli pyruvate dehydrogenase multienzyme complex: a mass spectrometric assay for reductive acetylation of dihydrolipoamide acetyltransferase. Protein Expr. Purif. 28:140-50 |
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| Abstract |
Plasmids were constructed for overexpression of the Escherichia coli dihydrolipoamide acetyltransferase (1-lip E2, with a single hybrid lipoyl domain per subunit) and dihydrolipoamide dehydrogenase (E3). A purification protocol is presented that yields homogeneous recombinant 1-lip E2 and E3 proteins. The hybrid lipoyl domain was also expressed independently. Masses of 45,953+/-73Da (1-lip E2), 50,528+/-5.5Da (apo-E3), 51,266+/-48Da (E3 including FAD), and 8982+/-4.0 (lipoyl domain) were determined by MALDI-TOF mass spectrometry. The purified 1-lip E2 and E3 proteins were functionally active according to the overall PDHc activity measurement. The lipoyl domain was fully acetylated after just 30 s of incubation with E1 and pyruvate. The mass of the acetylated lipoyl domain is 9019+/-2Da according to MALDI-TOF mass spectrometry. Treatment of the 1-lip E2 subunit with trypsin resulted in the appearance of the lipoyl domain with a mass of 10,112+/-3Da. When preincubated with E1 and pyruvate, this tryptic fragment was acetylated according to the mass increase. MALDI-TOF mass spectrometry was thus demonstrated to be a fast and precise method for studying the reductive acetylation of the recombinant 1-lip E2 subunit by E1 and pyruvate. |
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| Keywords |
Acetylation; Acetyltransferases/chemistry; Acetyltransferases/genetics; Acetyltransferases/isolation & purification; Acetyltransferases/metabolism; Amino Acid Sequence; Dihydrolipoamide Dehydrogenase/chemistry; Dihydrolipoamide Dehydrogenase/genetics; Dihydrolipoamide Dehydrogenase/isolation & purification; Dihydrolipoamide Dehydrogenase/metabolism; Dihydrolipoyllysine-Residue Acetyltransferase; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Gene Expression; Mass Spectrometry; Molecular Sequence Data; Oxidation-Reduction; Protein Subunits/chemistry; Protein Subunits/genetics; Protein Subunits/isolation & purification; Protein Subunits/metabolism; Pyruvate Dehydrogenase Complex/chemistry; Pyruvate Dehydrogenase Complex/genetics; Pyruvate Dehydrogenase Complex/isolation & purification; Pyruvate Dehydrogenase Complex/metabolism |
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