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

Citations Over TimeTop 10% of 1994 papers

Abstract

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)

Related Papers

• → Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion(2021)99 cited
• → α-Keto acid dehydrogenase complexes(1972)279 cited
• → Purification and properties of the pyruvate dehydrogenase complex from Salmonella typhimurium and formation of hybrids with the enzyme complex from escherichia coli(1982)17 cited
• → OXIDATION OF α-KETO ACIDS(1968)4 cited
• → REGULATION OF PYRUVATE DEHYDROGENASE BY PHOSPHORYLATION/DEPHOSPHORYLATION11Work supported by Medical Research Council (UK) and by the British Diabetic Association.(1979)4 cited