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 Enterococcus faecalis 10C1, E1alpha (bkdA), E1beta (bkdB), E2 (bkdC), and E3 (bkdD), were found to reside in the gene cluster ptb-buk-bkdDABC. The predicted products of ptb and buk exhibited significant homology to the phosphotransbutyrylase and butyrate kinase, respectively, from Clostridium acetobutylicum. Activity and redox properties of the purified recombinant enzyme encoded by bkdD indicate that E. faecalis has a lipoamide dehydrogenase that is distinct from the lipoamide dehydrogenase associated with the pyruvate dehydrogenase complex. Specific activity of the ptb gene product expressed in Escherichia coli was highest with the substrates valeryl-coenzyme A (CoA), isovaleryl-CoA, and isobutyryl-CoA. In cultures, a stoichiometric conversion of alpha-ketoisocaproate to isovalerate was observed, with a concomitant increase in biomass. We propose that alpha-ketoisocaproate is converted via the BKDH complex to isovaleryl-CoA and subsequently converted into isovalerate via the combined actions of the ptb and buk gene products with the concomitant phosphorylation of ADP. In contrast, an E. faecalis bkd mutant constructed by disruption of the bkdA gene did not benefit from having alpha-ketoisocaproate in the growth medium, and conversion to isovalerate was less than 2% of the wild-type conversion. It is concluded that the bkd gene cluster encodes the enzymes that constitute a catabolic pathway for branched-chain alpha-keto acids that was previously unidentified in E. faecalis.
Ward
D E
DE
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157, USA.
Ross
R P
RP
van der Weijden
C C
CC
Snoep
J L
JL
Claiborne
A
A
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000595:Amino Acid Sequence; D001483:Base Sequence; D003013:Clostridium / Q000201:enzymology; D004269:DNA, Bacterial; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics / Q000302:isolation & purification; D013293:Enterococcus faecalis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D007651:Keto Acids / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D005810:Multigene Family; D010084:Oxidation-Reduction; D010709:Phosphate Acetyltransferase / Q000235:genetics; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000235:genetics; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D007651:Keto Acids; D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D008058:Dihydrolipoamide Dehydrogenase; D010709:Phosphate Acetyltransferase; D017851:Phosphotransferases (Carboxyl Group Acceptor); C019285:butyrate kinase