Isolation and characterization of a complementary DNA clone coding for the E1.beta. subunit of the bovine branched-chain .alpha.-ketoacid dehydrogenase complex: complete amino acid sequence of the precursor protein and its proteolytic processing

Abstract

A 1.7-kb cDNA clone encoding the entire precursor of the E1.beta. subunit of the branched-chain .alpha.-ketoacid dehydrogenase (BCKDH) complex was isolated from a bovine liver cDNA library by screening with a mixture of synthetic oligonucleotide probes corresponding to the C-terminal five-residue sequence of the mature E1.beta. subunit. A partial amino acid sequence was determined by Edman degradation of the intact subunit and the peptides generated by cleavage at the lysyl bonds. Nucleotide sequences analysis revealed that the isolated cDNA clone contained the 5''-untranslated sequence of 186 nucleotides, the translated sequence of 1176 nucleotides, and the 3''-untranslated sequence of 306 nucleotides with a poly(A) tail. A type AATAAA polyadenylation signal was located 17 nucleotides upstream of the start of a poly(A) tail. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the partial amino acid sequence of the mature BCKDH E1.beta. subunit showed that the cDNA insert encodes for a 342 amino acid subunit with Mr 37,745 and that the subunit is synthesized as the precursor with a leader sequence of 50 amino acids and processed at the N-terminus. Northern blot analysis using the cDNA insert as a probe showed the presence of a 1.8-1.9-kb mRNA in bovine liver, suggesting that the insert covers nearly a full length of mRNA. Alignment of the deduced amino acid sequence of bovine BCKDH E1.beta. with that of the human pyruvate dehydrogenase (PDH) complex E1.beta. subunit revealed a high degree of sequence homology throughout the two enzymes. The structures and function of mammalian .alpha.-ketoacid dehydrogenase complexes are apparently highly conserved.