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 ac id decarboxylase decreased the CO2 production from alpha-keto-beta-met hylvalerate (KMV) suggesting the presence of an NADH dependent inhibit or in the crude enzyme from Bacillus subtilis. This NADH-dependent dec arboxylase inhibitor was purified to homogeneity. by a fast protein li quid 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 thr ee branched chain amino acids (BCAAs), valine, leucine, and isoleucine . The decarboxylase inhibitor was therefore identified as leucine dehy drogenase. A decreased substrate availability caused by leucine dehydr ogenase thus reasonably accounted for the NADH dependent inhibition of the decarboxylation. In turn, the observation that leucine dehydrogen ase 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 observa tion that addition of NAD to crude fatty acid synthetase increased the incorporation of isoleucine into BCFAs. Pyridoxal-5'-phosphate and al pha-ketoglutarate, cofactors for BCAA transaminase, modulated BCFA bio synthesis from isoleucine in vitro, suggesting also the involvement of transaminase reaction in BCFA biosynthesis.