Open AccessCloning and characterization of the Alcaligenes eutrophus 2‐oxoglutarate dehydrogenase complex
Author(s) -
Hein Silke,
Steinbüchel Alexander
Publication year - 1996
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1996.tb08054.x
Subject(s) - cloning (programming) , alcaligenes , biochemistry , chemistry , dehydrogenase , biology , bacteria , microbiology and biotechnology , genetics , enzyme , computer science , pseudomonas , programming language
Nucleotide sequence analysis of a 3.3‐kb genomic Eco RI fragment and of relevant subfragments of a genomic 13.2‐kb Sma I 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.