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 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 E1-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Maeng
C Y
CY
Biochemical Institute, University of Texas at Austin 78712.
Yazdi
M A
MA
Niu
X D
XD
Lee
H Y
HY
Reed
L J
LJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D017931:DNA Primers; C489060:PDX1 protein, S cerevisiae; D010455:Peptides; D011768:Pyruvate Dehydrogenase Complex; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D008058:Dihydrolipoamide Dehydrogenase