DIHYDROLIPOAMIDE DEHYDROGENASE-BINDING PROTEIN OF THE HUMAN PYRUVATE-DEHYDROGENASE COMPLEX - DNA-DERIVED AMINO-ACID-SEQUENCE, EXPRESSION, AND RECONSTITUTION OF THE PYRUVATE-DEHYDROGENASE COMPLEX

Protein X, recently renamed dihydrolipoamide dehydrogenase-binding pro tein (E3BP), is required for anchoring dihydrolipoamide dehydrogenase (E-3) to the dihydrolipoamide transacetylase (E-2) core of the pyruvat e dehydrogenase complexes of eukaryotes. DNA and deduced protein seque nces for E3BP 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 co mponent of the complex. The protein has 46% amino acid sequence identi ty in its amino-terminal region with the second lipoyl domain of E-2, 38% identity in its central region with the putative peripheral subuni t-binding domain of E-2, and 50% identity in its carboxyl-terminal reg ion with the catalytic inner care do. main of E-2. The similarity in t he latter domain stands in contrast to E3BP of Saccharomyces cerevisia e, 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 dihyrdrolipoamide acyltransferases is replac ed by a serine residue in human E3BP; thus, catalysis of coenzyme A ac etylation by this protein is unlikely, Coexpression of cDNAs for E3BP and E-2 resulted in the formation of ale E-2.E3BP subcomplex that spon taneously reconstituted the pyruvate dehydrogenase complex in the pres ence of native E-3 and recombinant pyruvate decarboxylase (E-1).