Cloning and characterization of theAlcaligenes eutrophus2-oxoglutarate dehydrogenase complex
Open Access
- 1 March 1996
- journal article
- Published by Oxford University Press (OUP) in FEMS Microbiology Letters
- Vol. 136 (3) , 231-238
- https://doi.org/10.1111/j.1574-6968.1996.tb08054.x
Abstract
Nucleotide sequence analysis of a 3.3-kb genomic EcoRI fragment and of relevant subfragments of a genomic 13.2-kb SmaI fragment of Alcaligenes eutrophus, which were identified by using a dihydrolipoamide dehydrogenase-specific DNA probe, revealed the structural genes of the 2-oxoglutarate dehydrogenase complex in a 7.5-kb genomic region. The genes odhA (2850 bp), odhB (1248 bp), and odhL (1422 bp), encoding 2-oxoglutarate dehydrogenase (El), dihydrolipoamide succinyltransferase (E2), and dihydrolipoamide dehydrogenase (E3), respectively, occur co-linearly in one gene cluster downstream of a putative −35 / −10 promoter in the order odhA, odhB, and odhL. In comparison to other bacteria, the occurrence of genes for two E3 components for the pyruvate as well as for the 2-oxoglutarate dehydrogenase complexes is unique. Heterologous expression of the A. eutrophus odh genes in E. coli XL1-Blue and in the kgdA 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.Keywords
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