In vivo incorporation of lipoic acid enantiomers and homologues in the pyruvate dehydrogenase complex from Escherichia coli.

The strain Escherichia coli JRG26, which has a defect in the lipoic acid biosynthesis, was cultivated in the presence of R-lipoic acid, S-lipoic acid, RS-dithiolane-3-caproic acid, RS-bisnorlipoic acid, and RS-tetranorlipoic acid, respectively. With the exception of the last compound the strain was able to grow with all these substances. R-lipoic acid was the most efficient factor, concentrations of 10 ng/l were sufficient to support growth of the cells, while 10(4)-fold to 10(7)-fold higher concentrations were necessary for the other compounds. The specific catalytic activity of the pyruvate dehydrogenase complex isolated from the cells grown on RS-dithiolane-3-caproic acid was only slightly lower than from cells grown on R-lipoic acid. With RS bisnorlipoic acid the specific activity was one third compared to that of the native enzyme complex. The incorporation of the RS-bisnorlipoic acid into the pyruvate dehydrogenase could directly be demonstrated by polyclonal antibodies directed against R-lipoic acid and RS-bisnorlipoic acid, both conjugated to BSA. Western blot analysis showed that the antibodies against the R-lipoic acid reacted specifically with the E2 component of pyruvate dehydrogenase complex purified from cells grown on this factor, while antibodies against RS-bisnorlipoic acid reacted with the enzyme complex isolated from cells grown in the presence of this compound.

Using lipoate enantiomers and thioredoxin to study the mechanism of the 2-oxoacid-dependent dihydrolipoate production by the 2-oxoacid dehydrogenase complexes.

The thioredoxin-catalyzed insulin reduction by dihydrolipoate was applied to study the 2-oxoacid: lipoate oxidoreductase activity of 2-oxoacid dehydrogenase complexes. The enzymatic and non-enzymatic mechanisms of the transfer of reducing equivalents from the complexes to free lipoic acid (alpha-lipoic acid, 6,8-thiooctic acid) were distinguished using the high stereoselectivity of the complex enzymes to the R-enantiomer of lipoate. Unlike these enzymes, thioredoxin from E. coli exhibited no stereoselectivity upon reduction with chemically obtained dihydrolipoate. However, coupled to the dihydrolipoate production by the dehydrogenase complexes, the process was essentially sensitive both to the enantiomer used and the dihydrolipoyl dehydrogenase activity of the complexes. These results indicated the involvement of the third complex component, dihydrolipoyl dehydrogenase, in the 2-oxoacid-dependent dihydrolipoate formation. The implication of the investigated reaction for a connection between thioredoxin and the 2-oxoacid dehydrogenase complexes in the mitochondrial metabolism are discussed.