cDNA cloning of the chicken branched-chain alpha-keto acid dehydrogenase complex - Chicken-specific residues of the acyltransferase affect the overall activity and the interaction with the dehydrogenase

Branched-chain alpha -keto acid dehydrogenase complex is a macromolecule co mprising three catalytic components: a dehydrogenase (E1) with alpha (2)bet a (2) structure, an acyltransferase (E2) and a dihydrolipoamide dehydrogena se (E3). In the mammalian complex, the E2 component with 24 identical subun its forms a structural core, to which multiple copies of E1 and E3 bind non covalently. We isolated cDNA clones encoding E1 alpha, E1 beta and E2 subun its from a chicken-liver cDNA library and performed nucleotide sequencing. Amino-acid sequences deduced from the nucleotide sequences revealed that ch icken E1 alpha and E1 beta chains had substantially homologous sequences wi th the corresponding mammalian polypeptides, except for the N-terminus. Chi cken E2 conserved three functional domains, a lipoyl-bearing domain, an E1/ E3 binding domain and an inner-core domain, but contrasted strongly with ma mmalian E2 in respect of containing 11 additional residues in two interdoma in linkers: nine sequential residues in one linker and two residues in the other. Replacement of many residues was also observed in the chicken linker s. When E2 activity for catalyzing the overall reaction was measured by act ivity reconstitution in combination with E1 and E3, chicken E2 was markedly less effective than mammalian E2. The capability of chicken E2 for binding E1 was also reduced when determined by the binding assay using sucrose den sity gradient centrifugation. Chicken E1 was functionally as well as struct urally indistinguishable from mammalian E1. Thus the reduced catalytic acti vity of chicken E2 must arise from its reduced E1-binding capacity, which r esults from the characteristic structure of interdomain linkers in chicken E2.