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 comprising three catalytic components: a dehydrogenase (E1) with alpha(2)beta(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 E1 alpha, E1 beta and E2 subunits from a chicken-liver cDNA library and performed nucleotide sequencing. Amino-acid sequences deduced from the nucleotide sequences revealed that chicken E1 alpha and E1 beta chains had substantially homologous sequences with the corresponding mammalian polypeptides, except for the N-terminus. Chicken E2 conserved three functional domains, a lipoyl-bearing domain, an E1/E3 binding domain and an inner-core 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.
Ono
K
K
Department of Biochemistry, Fukushima Medical University, Japan.
Hakozaki
M
M
Suzuki
T
T
Mori
T
T
Hata
H
H
Kochi
H
H
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002645:Chickens; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000378:metabolism*; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005260:Female; D015723:Gene Library; D006801:Humans; D015151:Immunoblotting; D007658:Ketone Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D009097:Multienzyme Complexes; D015345:Oligonucleotide Probes; D010455:Peptides; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D008058:Dihydrolipoamide Dehydrogenase; D000217:Acyltransferases