NADH-dependent inhibition of branched-chain fatty acid synthesis in Bacillus subtilis.
Addition of NADH to crude but not to pure branched-chain alpha-keto acid decarboxylase decreased the CO2 production from alpha-keto-beta-methylvalerate (KMV) suggesting the presence of an NADH dependent inhibitor in the crude enzyme from Bacillus subtilis. This NADH-dependent decarboxylase inhibitor was purified to homogeneity by a fast protein liquid chromatography system. The purified inhibitor was identical with leucine dehydrogenase as to N-terminal amino acid squence (35 residues) and molecular weight, and catalyzed the oxidative deamination of three branched chain amino acids (BCAAs), valine, leucine, and isoleucine. The decarboxylase inhibitor was therefore identified as leucine dehydrogenase. A decreased substrate availability caused by leucine dehydrogenase thus reasonably accounted for the NADH dependent inhibition of the decarboxylation. In turn, the observation that leucine dehydrogenase competes with the decarboxylase for branched-chain alpha-keto acid (BCKA) suggested an involvement of this enzyme in the branched chain fatty acid (BCFA) biosynthesis. This view was supported by the observation that addition of NAD to crude fatty acid synthetase increased the incorporation of isoleucine into BCFAs. Pyridoxal-5'-phosphate and alpha-ketoglutarate, cofactors for BCAA transaminase, modulated BCFA biosynthesis from isoleucine in vitro, suggesting also the involvement of transaminase reaction in BCFA biosynthesis.
Oku
H
H
Laboratory of Applied Biochemistry, Faculty of Agriculture, University of The Ryukyus, Okinawa-Ken, Japan.
Fujita
K
K
Nomoto
T
T
Suzuki
K
K
Iwasaki
H
H
Chinen
I
I
D000217:Acyltransferases / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors; D003065:Coenzyme A / Q000378:metabolism; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000096:biosynthesis*; D007700:Kinetics; D050803:Leucine Dehydrogenase; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009243:NAD / Q000378:metabolism*
D003470:Culture Media; D005227:Fatty Acids; D009097:Multienzyme Complexes; D009243:NAD; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase; D000217:Acyltransferases; C057800:branched-chain fatty acid synthetase; D002262:Carboxy-Lyases; D003065:Coenzyme A