Cloning and sequence analysis of the genes encoding the α and β subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

Abstract

A 4175-bp EcoRI fragment of DNA that encodes the .alpha. and .beta. chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escheria coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the E1 .alpha. subunit (368 amino acids, Mr 41312, without the initiating methionine residue) and E1.beta. subunit (324 amino acids, Mr 35306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1.beta. gene begins just 3 bp downstream from the E1 .alpha. stop codon. It is followed, after a longer gap of 73bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The E1 .alpha. and E1.beta. subunits of the B. stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1.alpha. and E1.beta. subunits of pyruvate and branched-chain 2-oxo-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the E1.alpha. chain contains the highly conserved motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.