Expression, Purification, and Characterization of the Dihydrolipoamide Dehydrogenase-Binding Protein of the Pyruvate Dehydrogenase Complex from Saccharomyces cerevisiae

Genes encoding dihydrolipoamide dehydrogenase (E3) and the E3-binding protein (E3BP, protein X), components of the Saccharomyces cerevisiae pyruvate dehydrogenase (PDH) complex, were coexpressed in Escherichia coli to produce an E3BP-E3 complex, thereby minimizing proteolysis of E3BP and facilitatin...

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Bibliographic Details
Published in:Biochemistry (Easton) 1994-11, Vol.33 (46), p.13801-13807
Main Authors: Maeng, Cheol-Young, Yazdi, Mohammad A, Niu, Xiao-Da, Lee, Hoi Y, Reed, Lester J
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
Dihydrolipoamide Dehydrogenase - genetics
Dihydrolipoamide Dehydrogenase - metabolism
DNA Primers
Escherichia coli
expresion genica
expression des genes
gene expression
Genetic Vectors
Molecular Sequence Data
oxidoreductases
oxidorreductasas
oxydoreductase
Peptides - genetics
Peptides - isolation & purification
Peptides - metabolism
purificacion
purification
Pyruvate Dehydrogenase Complex - genetics
Pyruvate Dehydrogenase Complex - isolation & purification
Pyruvate Dehydrogenase Complex - metabolism
Recombinant Fusion Proteins
saccharomyces cerevisiae
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins
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Summary:Genes encoding dihydrolipoamide dehydrogenase (E3) and the E3-binding protein (E3BP, protein X), components of the Saccharomyces cerevisiae pyruvate dehydrogenase (PDH) complex, were coexpressed in Escherichia coli to produce an E3BP-E3 complex, thereby minimizing proteolysis of E3BP and facilitating its purification. The 2 genes were linked into a single transcriptional unit separated by a 31-nucleotide segment containing a ribosome-binding sequence. The E3BP-E3 complex was highly purified and then separated into E3 and E3BP by chromatography on hydroxylapatite in the presence of 5 M urea. The E3BP-E3 complex combined rapidly with a pyruvate dehydrogenase (E1)-dihydrolipoamide acetyltransferase (E2) subcomplex (E1-E2 subcomplex) to reconstitute a functional PDH complex, with pyruvate oxidation activity similar to that of PDH complex from bakers' yeast. The stoichiometry of binding of E3BP and E3BP-E3 complex to the 60-subunit pentagonal dodecahedron-like E2 was determined with a truncated form of E2 (tE2, residues 206-454) lacking the lipoyl domain and the El-binding domain, and with E1-E2 subcomplex, which contains intact E2. Mixtures containing tE2 or E1-E2 subcomplex and excess E3BP or E3BP-E3 complex were subjected to ultracentrifugation to separate the large complexes from unbound E3BP or E3BP-E3, and the complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer. The results showed that the E1-E2 subcomplex binds about 12 E3BP monomers attached to 12 E3 homodimers. Similar results were obtained by analysis of highly purified PDH complex from bakers' yeast. Somewhat more E3BP (approximately 15 molecules) and E3BP-E3 complex (approximately 14 molecules) bound to tE2. Structural considerations suggest that 1 E3BP molecule, bearing an E3 homodimer, is bound in each of the 12 faces of the pentagonal dodecahedron-like E2.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00250a034