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.

Ten mutants of the yeast

Squalene epoxidase is an essential enzyme in the ergosterol-biosynthesis pathway. It catalyzes the epoxidation of squalene to 2,3-oxidosqualene and is the specific target of the antifungal drug terbinafine. Treatment of yeast cells with this inhibitor leads to squalene accumulation and sterol depletion. As ergosterol fulfils several essential functions, each requiring optimal sterol concentrations, synthesis of sterols in yeast must be tightly regulated. This study focuses on the sterol-mediated regulation of expression of the ERG1 gene, which codes for squalene epoxidase in Saccharomyces cerevisiae. Inhibition of ergosterol biosynthesis with terbinafine increases the expression of ERG1 in a concentration-dependent manner to a maximum of sevenfold. Inhibition of later steps in the ergosterolbiosynthetic pathway by ketoconazol, an inhibitor of the lanosterol-14a-demethylase, and U18666A, an inhibitor of the squalene-2,3-epoxide±lanosterol cyclase, also induce expression of ERG1, suggesting that ERG1 expression is positively regulated by diminished intracellular ergosterol levels. The regulatory effect of sterols is manifested at the level of transcription. Deletion analysis of the ERG1 promoter identified a novel regulatory DNA sequence element. Two 6-bp direct repeats, separated by 4 bp, AGCTCGGCCGAGCTCG, are unique to the ERG1 promoter. A DNA fragment containing this region confers ergosterol-regulated expression on an otherwise unregulated CYC1 promoter construction. One copy of the 6-bp element, AGCTCG, is sufficient to confer regulation, albeit less effectively than when both elements are present, whereas the removal of both elements from the ERG1 promoter leads to the loss of sterol-dependent ERG1 regulation.

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