CLONING AND CHARACTERIZATION OF THE ALCALIGENES-EUTROPHUS 2-OXOGLUTARATE DEHYDROGENASE COMPLEX

Nucleotide sequence analysis of a 3.3-kb genomic EcoRI fragment and of relevant subfragments of a genomic 13.2-kb SmaI fragment of Alcaligen es eutrophus, which were identified by using a dihydrolipoamide dehydr ogenase-specific DNA probe, revealed the structural genes of the 2-oxo glutarate dehydrogenase complex in a 7.5-kb genomic region. The genes odhA (2850 bp), odhB (1248 bp), and odhL (1422 bp), encoding 2-oxoglut arate dehydrogenase (El), dihydrolipoamide succinyltransferase (E2), a nd dihydrolipoamide dehydrogenase (E3), respectively, occur co-linearl y in one gene cluster downstream of a putative -35/-10 promoter in the order odhA, odhB, and odhL. In comparison to other bacteria, the occu rrence of genes for two E3 components for the pyruvate as well as for the 2-oxoglutarate dehydrogenase complexes is unique. Heterologous exp ression of the A. eutrophus odh genes in E. coli XL1-Blue and in the k gdA mutant Pseudomonas putida JS347 was demonstrated by the occurrence of protein bands in electropherograms, by spectrometric detection of enzyme activities, and by phenotypic complementation, respectively.