Catabolism of branched-chain alpha-keto acids in Enterococcus faecalis: the bkd gene cluster, enzymes, and metabolic route

Genes encoding a branched-chain alpha-keto acid dehydrogenase from Enteroco ccus faecalis 10C1, E1 alpha (bkdA), E1 beta (bkdB), E2 (bkdC, and E3 (bkdD ), were found to reside in the gene cluster ptb-bllk-bkdDABC.The predicted products of ptb and buk exhibited significant homology to the phosphotransb utyrylase and butyrate kinase, respectively, from Clostridium acetobutylicu m. Activity and redox properties of the purified recombinant enzyme encoded by bkdD indicate that E. faecalis has a lipoamide dehydrogenase that is di stinct from the lipoamide dehydrogenase associated with the pyruvate dehydr ogenase complex. Specific activity of the ptb gene product expressed in Esc herichia coli was highest with the substrates valeryl coenzyme A (CoA), iso valeryl-CoA, and isobutyryl-CoA. In cultures, a stoichiometric conversion o f alpha-ketoisocaproate to isovalerate was observed, with a concomitant inc rease in biomass. We propose that alpha-ketoisocaproate is converted via th e BKDH complex to isovaleryl-CoA and subsequently converted into isovalerat e via the combined actions of the ptb and buk gene products with the concom itant phosphorylation of ADP. In contrast, an E. faecalis bkd mutant constr ucted by disruption of the bkdA gene did not benefit from having alpha-keto isocaproate in the growth medium, and conversion to isovalerate was less th an 2% of the wild-type conversion. It is concluded that the bkd gene cluste r encodes the enzymes that constitute a catabolic pathway for branched-chai n alpha-keto acids that was previously unidentified in E. faecalis.