Dihydrolipoamide acyltransferase (E 2 ), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of ␣-keto acids, forms the central core to which the other components attach. We have determined the structures of the truncated 60-mer core dihydrolipoamide acetyltransferase (tE 2 ) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex and complexes of the tE 2 core associated with a truncated binding protein (tBP), intact binding protein (BP), and the BP associated with its dihydrolipoamide dehydrogenase (BP⅐E 3 ). The tE 2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges. Previous studies have given rise to the generally accepted belief that the other components are bound on the outside of the E 2 scaffold. However, this investigation shows that the 12 large openings in the tE 2 core permit the entrance of tBP, BP, and BP⅐E 3 into a large central cavity where the BP component apparently binds near the tip of the tE 2 trimer. The bone-shaped E 3 molecule is anchored inside the central cavity through its interaction with BP. One end of E 3 has its catalytic site within the surface of the scaffold for interaction with other external catalytic domains. Though tE 2 has 60 potential binding sites, it binds only about 30 copies of tBP, 15 of BP, and 12 of BP⅐E 3 . Thus, E 2 is unusual in that the stoichiometry and arrangement of the tBP, BP, and E 3 ⅐BP components are determined by the geometric constraints of the underlying scaffold.Pyruvate dehydrogenase complexes (PDCs) 1 are among the largest (M r ϳ10 6 -10 7 ) and most complex multienzyme structures known. They consist of a central core that has both functional and structural roles in organizing the complex, the dihydrolipoamide acetyltransferase (E 2 ) subunits associate to form the core complex that also serves as a scaffold to which the other components are attached (1-4). Electron microscopy (5-8) and x-ray crystallography (9 -11) studies have revealed two fundamental morphologies of the E 2 cores. The cubic E 2 core of the Escherichia coli PDC has 24 subunits arranged with octahedral symmetry, whereas the pentagonal dodecahedral E 2 core of the PDC complexes from eukaryotes and some Grampositive bacteria has 60 subunits arranged with icosahedral symmetry. The subunits form cone-shaped trimers at each of the 8 and 20 vertices of the cubic and dodecahedral structures, respectively. These trimers are interconnected by bridges to form a cage-like complex (8 -11).The E 2 subunit is a multidomain structure to which the other constituents of the functional PDC (1-4) bind (see Fig. 1). These include the pyruvate dehydrogenase (E 1 ) and dihydrolipoamide dehydrogenase (E 3 ). E 3 requires a binding protein (BP) to anchor it to the core of the yeast (12) and mammalian PDCs (13-15) though, in E. coli and Bacillus stearothermophilus PDCs, BP is not required (1-4).It is widely held that the constituent proteins are bound to the out...
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)
A Baciffus subtifis gerC spore germination mutant demonstrating a temperature-sensitive response to L-alanine as germinant has been characterized in detail. The gerC58 mutation is 50% cotransformed with aroB in the gene order gerC-aroRtrpC. The mutation is responsible for a severe growth defect which is manifest at all growth temperatures and is most extreme on rich media. A second, unlinked, mutation in the original strain suppressed this growth defect, but spores of the suppressed strain failed to germinate in alanine at 42 "C. As this germination defect is dependent on the presence of the gerC58 allele, it is likely to be the direct result of a mutant gerC protein. The gerC gene therefore appears to have a role in both spore gemination and vegetative cell growth. A gene library of Bcfbdigested B. subtifis chromosomal DNA was constructed in phage vector &105527. A derivative containing the gerC region was obtained by complementation of the growth defect of an unsuppressed gerC58 strain. This phage contained a 6.3 kb insert of bacterial DNA, which is above the reported packaging limit of the phage. It failed to form visible plaques, although it could be handled as a prophage and sufficient phage particles be isolated to allow characterization of the insert. A deletion derivative generated in vitro and carrying only 2.9 kb of insert DNA also complemented the gerC defect. This gerC locus is the second locus to be implicated in alanine-stimulated germination. The first, gerA, is a developmentally controlled operon whose gene products are present only in the spore. This study of g e e , in contrast, reveals a role in spore germination for a normally essential vegetative protein.
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