The 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii. 2. Molecular cloning and sequence analysis of the gene encoding the succinyltransferase component.

The nucleotide sequence encoding the succinyltransferase component (E2o) of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined. Previously the cloning in Escherichia coli of the gene encoding lipoamide dehydrogenase from A. vinelandii was reported [Westphal, A.H. & de Kok, A. (1988) Eur. J. Biochem. 172, 299-305]. The 3.2-kb fragment used for the sequence determination contained the main part of the gene encoding succinyltransferase. The complete E2o gene, as well as the gene encoding the 2-oxoglutarate dehydrogenase component, resided on a 14.7-kb fragment from which the 3.2-kb fragment was subcloned. The protein-coding sequence of the gene consists of 1200 bp (400 codons, including the AUG start codon and the UGA stop codon). It is separated from the gene encoding the 2-oxoglutarate dehydrogenase component by 42 bp. No E. coli-like promoter sequence was found. A putative ribosome-binding site is located 9-15 bp upstream from the start codon. No terminator sequences were found downstream of the stop codon. This makes it likely that the three genes of the oxoglutarate dehydrogenase complex are transcribed as a single mRNA transcript analogous to the pyruvate dehydrogenase complex in E. coli. The intact gene was subcloned from the 14.7-kb fragment and brought to high expression under the influence of the vector-encoded lacZ promoter. The similarity with the E. coli enzyme is high with 63% identity. Like the enzyme from E. coli, it consists of a single lipoyl-binding domain, a putative E1- and E3-binding domain and a catalytic domain. The main difference is found in a 31-residue sequence rich in alanine and proline located between the lipoyl domain and the putative E1- and E3-binding domain. This sequence, usually found in acetyltransferases and there identified as a highly mobile region by 1H-NMR, is replaced by a more polar, charged region in the E. coli enzyme.