Dihydrolipoamide dehydrogenases of Advenella mimigardefordensis and Ralstonia eutropha catalyze cleavage of 3,3'-dithiodipropionic acid into 3-mercaptopropionic acid.
The catabolism of the disulfide 3,3'-dithiodipropionic acid (DTDP) is initiated by the reduction of its disulfide bond. Three independent Tn5::mob-induced mutants of Advenella mimigardefordensis strain DPN7(T) were isolated that had lost the ability to utilize DTDP as the sole source of carbon and energy and that harbored the transposon insertions in three different sites of the same dihydrolipoamide dehydrogenase gene encoding the E3 subunit of the pyruvate dehydrogenase multi-enzyme complex of this bacterium (LpdA(Am)). LpdA(Am) was analyzed in silico and compared to homologous proteins, thereby revealing high similarities to the orthologue in Ralstonia eutropha H16 (PdhL(Re)). Both bacteria are able to cleave DTDP into two molecules of 3-mercaptopropionic acid (3MP). A. mimigardefordensis DPN7(T) converted 3MP to 3-sulfinopropionic acid, whereas R. eutropha H16 showed no growth with DTDP as the sole carbon source but was instead capable of synthesizing heteropolythioesters using the resulting cleavage product 3MP. Subsequently, the genes lpdA(Am) and pdhL(Re) were cloned, heterologously expressed in Escherichia coli applying the pET23a expression system, purified, and assayed by monitoring the oxidation of NADH. The physiological substrate lipoamide was reduced to dihydrolipoamide with specific activities of 1,833 mkat/kg of protein (LpdA(Am)) or 1,667 mkat/kg of protein (PdhL(Re)). Reduction of DTDP was also unequivocally detected with the purified enzymes, although the specific enzyme activities were much lower: 0.7 and 0.5 mkat/kg protein, respectively.
Wübbeler
Jan Hendrik
JH
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany.
Raberg
Matthias
M
Brandt
Ulrike
U
Steinbüchel
Alexander
A
D015097:3-Mercaptopropionic Acid / Q000378:metabolism*; D042401:Alcaligenaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D005798:Genes, Bacterial; D008623:Mercaptoethanol / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011422:Propionates / Q000378:metabolism*
D004269:DNA, Bacterial; D004220:Disulfides; D011422:Propionates; D008623:Mercaptoethanol; D015097:3-Mercaptopropionic Acid; D008058:Dihydrolipoamide Dehydrogenase