THE ENOYL-[ACYL-CARRIER-PROTEIN] REDUCTASE (FABI) OF ESCHERICHIA-COLI, WHICH CATALYZES A KEY REGULATORY STEP IN FATTY-ACID BIOSYNTHESIS, ACCEPTS NADH AND NADPH AS COFACTORS AND IS INHIBITED BY PALMITOYL-COA

Reduction of enoyl-acyl-carrier-protein (ACP) substrates by enoyl-ACP reductase is a key regulatory step in fatty acid elongation of Escheri chia coli. Two enoyl-ACP reductase activities have been described in E . coli, one specific for NADH, the other for NADPH as cofactor. Becaus e of their distinct enzymatic properties, these activities were ascrib ed to two different proteins. The NADH-dependent enoyl-ACP reductase o f E. coli has previously been identified as the FabI protein, which is the target of a group of antibacterial compounds, the diazaborines. W e now demonstrate that both enoyl-ACP reductase activities reside in F abI. In crude cell extracts of FabI-overproducing strains, both NADH-d ependent and NADPH-dependent enoyl-ACP reductase activities are increa sed. Mutations in the fabI gene that lead either to temperature-sensit ive growth or diazaborine resistance result in the reduction of both a ctivities. When FabI is purified in pH 6.5 buffers, the protein exhibi ts NADH-dependent and NADPH-dependent reductase activities. Both enzym atic activities are inhibited by diazaborine. The NADPH-dependent enoy l-ACP reductase activity, however, turned out to be approximately eigh t times more resistant to diazaborine. The difference in sensitivity i ndicates that binding of either NADPH or NADH to FabI results in disti nct changes in the configuration of the protein or, alternatively, it is different due to the different charge of the cofactors. These effec ts might be responsible for the differences in the enzymatic propertie s. Both reductase activities of the FabI protein are inhibited by phys iologically relevant concentrations of palmitoyl-CoA, which might be i mportant in regulating endogenous fatty acid biosynthesis in E. coli i n the presence of exogenous fatty acids.