cDNA cloning of the chicken branched‐chain α‐keto acid dehydrogenase complex

Abstract

Branched-chain a-keto acid dehydrogenase complex is a macromolecule comprising three catalytic components: a dehydrogenase (E1) with a 2 b 2 structure, an acyltransferase (E2) and a dihydrolipoamide dehydrogenase (E3). In the mammalian complex, the E2 component with 24 identical subunits forms a structural core, to which multiple copies of E1 and E3 bind noncovalently. We isolated cDNA clones encoding E1a, E1b and E2 subunits from a chicken-liver cDNA library and performed nucleotide sequencing. Amino-acid sequences deduced from the nucleotide sequences revealed that chicken E1a and E1b chains had substantially homologous sequences with the corresponding mammalian polypeptides, except for the N-terminus. Chicken E2 conserved three functional domains, a lipoylbearing domain, an E1/E3 binding domain and an innercore domain, but contrasted strongly with mammalian E2 in respect of containing 11 additional residues in two interdomain linkers: nine sequential residues in one linker and two residues in the other. Replacement of many residues was also observed in the chicken linkers. When E2 activity for catalyzing the overall reaction was measured by activity 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 density gradient centrifugation. Chicken E1 was functionally as well as structurally indistinguishable from mammalian E1. Thus the reduced catalytic activity of chicken E2 must arise from its reduced E1-binding capacity, which results from the characteristic structure of interdomain linkers in chicken E2.

Description of the mutations in 15 subjects with variant forms of maple syrup urine disease

“…None of the novel mutations was registered as a non-synonymous coding single-nucleotide polymorphism. Most variations described here affect highly conserved residues between the human E1 or E2 component and their homologous proteins as compared in the nucleotide databases from bacterial (Pseudomonas putida) and animal (Bos taurus, Rattus norvegicus, Gallus gallus) genomes (Aevarsson et al 2000;Ono et al 2001), strengthening their impact on the structure/function of the proteins. In addition, the disease-causing effect was assumed when the alteration led to a premature termination codon.…”

Interaction of peroxiredoxin V with dihydrolipoamide branched chain transacylase E2 (DBT) in mouse kidney under hypoxia

“…In particular, DBT belongs to the transacylase (E2) subunit and is a key enzyme in amino acid metabolism. Each E2 subunit consists of three independently functional domains: a lipoyl-bearing domain located in the N-terminal portion, an E1/E3-binding domain, and an inner-core domain at the C-terminal portion, with the three domains tethered by flexible linker regions [ 20 ]. The core domains of E2 subunit form a 24-meric scaffold, which is decorated with multiple copies of E1 and E3 attached through the subunit-binding domain [ 19 ].…”