Dihydrolipoamide Dehydrogenase-binding Protein of the Human Pyruvate Dehydrogenase Complex
Journal of Biological Chemistry
Protein X, recently renamed dihydrolipoamide dehydrogenase-binding protein (E 3 BP), is required for anchoring dihydrolipoamide dehydrogenase (E 3 ) to the dihydrolipoamide transacetylase (E 2 ) core of the pyruvate dehydrogenase complexes of eukaryotes. DNA and deduced protein sequences for E 3 BP of the human pyruvate dehydrogenase complex are reported here. With the exception of only a single lipoyl domain, the protein has a segmented multi-domain structure analogous to that of the E 2
... ent of the complex. The protein has 46% amino acid sequence identity in its amino-terminal region with the second lipoyl domain of E 2 , 38% identity in its central region with the putative peripheral subunit-binding domain of E 2 , and 50% identity in its carboxyl-terminal region with the catalytic inner core domain of E 2 . The similarity in the latter domain stands in contrast to E 3 BP of Saccharomyces cerevisiae, which is quite different from its homologous transacetylase in this region. The putative catalytic site histidine residue present in the inner core domains of all dihydrolipoamide acyltransferases is replaced by a serine residue in human E 3 BP; thus, catalysis of coenzyme A acetylation by this protein is unlikely. Coexpression of cDNAs for E 3 BP and E 2 resulted in the formation of an E 2 ⅐E 3 BP subcomplex that spontaneously reconstituted the pyruvate dehydrogenase complex in the presence of native E 3 and recombinant pyruvate decarboxylase (E 1 ). The pyruvate dehydrogenase complex (PDC) 1 catalyzes the oxidative decarboxylation of pyruvate with the formation of CO 2 , acetyl-CoA, and NADH. The eukaryotic complex has 30 copies of a tetrameric (␣ 2 ␤ 2 ) pyruvate decarboxylase (E 1 ) component noncovalently bound along the edges of an icosahedral 60 meric dihydrolipoamide acetyltransferase (E 2 ) core (1). Twelve copies of a homodimeric dihydrolipoamide dehydrogenase (E 3 ) component are believed held on the faces of the E 2 core by a corresponding number of monomeric E 3 -binding proteins (protein X, E 3 BP) (2, 3). With the exception of E 3 BP, the role of each enzymatic component in the overall reaction catalyzed by the complex is basically understood. E 1 catalyzes a thiamine diphosphate-dependent oxidative decarboxylation of pyruvate and the reductive acetylation of a lipoyl residue covalently attached to the lipoyl domain of E 2 . E 2 then catalyzes transfer of the acetyl group to coenzyme A, leaving a reduced E 2 lipoyl group that the E 3 component uses as an electron source for FAD-dependent reduction of NAD ϩ to NADH. Called protein X because its function was not apparent at the time it was originally discovered to be a component of eukaryotic PDCs (4 -6), E 3 BP has generated considerable interest because of sequence similarity with E 2 (7-10) and evidence that it has a covalently bound lipoyl moiety (4, 6, 8, 11) . Its function was difficult to establish in early studies because of a very tight association with the E 2 core of the complex. Very elegant limited proteolysis and immunological studies (12, 13) provided the first evidence that protein X contributes to the binding and function of E 3 . Cloning of the E 3 BP gene of Saccharomyces cerevisiae (14) revealed a protein structure that resembles the E 2 component of yeast in its amino terminus but not the remainder of the molecule. Subsequent gene disruption studies provided definitive proof that protein X should be considered an E 3 -binding protein (15). Questions left unsettled included whether mammalian E 3 BP is completely analogous to yeast E 3 BP, whether the lipoyl moiety of mammalian E 3 BP is functionally important for catalytic activity of the complex, and whether the inner core of the mammalian E 3 BP has transacetylase activity. The deduced amino acid sequence for the first mammalian (human) E 3 BP is presented here. Although similar in their amino-terminal, lipoyl-bearing domains, the mammalian E 3 BP and the yeast E 3 BP are markedly different in their carboxylterminal regions. Indeed, the human E 3 BP is more homologous to mammalian E 2 throughout its primary sequence than it is to yeast E 3 BP. Previous evidence that the mammalian E 3 BP contains only a single lipoyl domain in its amino terminus (8), rather than the two tandemly arranged lipoyl domains characteristic of mammalian E 2 , is confirmed. The active site histidine residue characteristic of transacetylases of all ␣-ketoacid dehydrogenase complexes is not conserved in the mammalian E 3 BP.