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

Abstract

Reduction of enoyl -acyl-carrier-protein (ACP) substrates by enoyl-ACP reductase is a key regulatory step in fatty acid elongation of Escherichia coli. Two enoyl-ACP reductase activities have been described in E. coli, one specific for NADH, the other for NADPH as cofactor. Because of their distinct enzymatic properties, these activities were ascribed to two different proteins. The NADH-dependent enoyl-ACP reductase of E. coli has previously been identified as the FabI protein, which is the target of a group of antibacterial compounds, the diazaborines. We now demonstrate that both enoyl-ACP reductase activities reside in FabI. In crude cell extracts of FabI-overproducing strains, both NADH-dependent and NADPHdependent enoyl-ACP reductase activities are increased. Mutations in the fabl gene that lead either to temperature-sensitive growth or diazaborine resistance result in the reduction of both activities. When FabI is purified in pH 6.5 buffers, the protein exhibits NADH-dependent and NADPH-dependent reductase activities. Both enzymatic activities are inhibited by diazaborine. The NADPH-dependent enoyl-ACP reductase activity, however, turned out to be approximately eight times more resistant to diazaborine. The difference in sensitivity indicates that binding of either NADPH or NADH to FabI results in distinct changes in the configuration of the protein or, alternatively, it is different due to the different charge of the cofactors. These effects might be responsible for the differences in the enzymatic properties. Both reductase activities of the FabI protein are inhibited by physiologically relevant concentrations of palmitoyl-CoA, which might be important in regulating endogenous fatty acid biosynthesis in E. coli in the presence of exogenous fatty acids.

Kinetic mechanism of NADH‐enoyl‐ACP reductase from Brassica napus

“…These data con¢rm that the B. napus cDNA encodes an NADH-speci¢c ENR and in this respect it is unlike the E. coli enzyme that is able to utilise both NADH and NADPH as cofactor [17].…”

Large D/H variations in bacterial lipids reflect central metabolic pathways

“…Differing fractionations between the enzyme types are therefore plausible. However, E. coli has only 1 of each reductase enzyme (i.e., a single FabG and FabI) yet lipid/water fractionations still vary substantially (23,37,42,43). C. necator has several putative ␤-ketoacyl ACP reductases and FabI-type enoyl ACP reductases (26), but fractionations in C. necator are similar to those of E. coli.…”

Crystal Structure of the Mycobacterium tuberculosis Enoyl-ACP Reductase, InhA, in Complex with NAD+ and a C16 Fatty Acyl Substrate

“…More recently, isoniazid was shown to prevent radiolabel from being incorporated into the chain extension of C24 -C26 fatty acyl substrates (16 -20), suggesting that the target of isoniazid action is within the mycobacterial FAS-II system. Using a genetic approach to isolate the isoniazid target, a single open reading frame was identified (referred to as inhA) in which transformation of the wild-type gene carried on a multicopy plasmid or allelic exchange with a single amino acid substitution mutant (Ser 94 to Ala) was sufficient to confer isoniazid resistance in Mycobacterium smegmatis (21).The protein encoded by the inhA gene, referred to as InhA, has a similar amino acid sequence to two previously characterized enoyl-ACP reductases, namely FabI from E. coli (28% identity) (22)(23)(24)(25), and ENR 1 from Brassica napus (oilseed rape) (23% identity) (26,27). Further analysis revealed that InhA catalyzes the NADH-dependent reduction of the trans double bond between positions C2 and C3 of fatty acyl substrates (28).…”

“…The protein encoded by the inhA gene, referred to as InhA, has a similar amino acid sequence to two previously characterized enoyl-ACP reductases, namely FabI from E. coli (28% identity) (22)(23)(24)(25), and ENR 1 from Brassica napus (oilseed rape) (23% identity) (26,27). Further analysis revealed that InhA catalyzes the NADH-dependent reduction of the trans double bond between positions C2 and C3 of fatty acyl substrates (28).…”