Enoyl acyl carrier protein (ACP) reductase (FabI) is a potential target for the development of antibacterial agents. Three-dimensional quantitative structure-activity relationships (3D-QSAR) for substituted formamides series of FabI inhibitors were investigated using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques. Pharmacophore and molecular docking methods were used for construction of the molecular alignments. A training set of 36 compounds was performed to create the 3D-QSAR models and their external predictivity was proven using a test set of 11 compounds. Graphical interpretation of the results revealed important structural features of the formamides related to the active site of FabI. The results may be exploited for further optimization of the design of new potent FabI inhibitors.

Transcarboxylase (TC) is a biotin-containing enzyme catalyzing the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate to form propionyl-CoA and oxalacetate. The transfer is achieved via carboxylated biotin bound to a 1.3S subunit within the multisubunit enzyme complex. The 1.3S subunit of TC is a 123 amino acid polypeptide, to which biotin is covalently attached at Lys 89. We have overexpressed 1.3S in Escherichia coli and characterized the biotinylated and apo-forms by 1D- and 2D-NMR spectroscopy. To search for protein-biotin interactions, which could modulate the reactivity of the biotin ring on the 1.3S subunit, we have compared the chemical shifts, relaxation parameters, and NH exchange rates of the ureido ring protons of free and 1.3S-bound biotin. These properties are similar for both forms of the biotin. Further, NOE experiments on 1.3S revealed no detectable cross peaks between biotin and the protein. Consistent with these findings, the 2D NMR data for holo- and apo-1.3S are essentially identical indicating little or no changes in conformation between the two forms of the protein. The conclusion that strong protein-biotin interactions do not exist in 1.3S contrasts with the findings for the biotin carboxylase carrier protein from E. coli acetyl-CoA carboxylase, which reveal significant biotin-protein contacts [Athappilly, F. K., and Hendrickson, W. A. (1995) Structure 3, 1407-1419]. Further, the biotin NH1\' exchange rates determined for 1.3S show that in the region of optimal activity for TC (pH 5.5-6.5) acid-catalyzed exchange predominates. In this pH range the base-catalyzed rate is too small (< 1 s-1) to account for the turnover rate of the enzyme. Thus, the means by which the N1\' atom is activated for nucleophilic attack of the carboxyl group in methylmalonyl-CoA does not appear to depend on interactions within the 1.3S subunit alone; rather activation must occur at the interfaces of the subunits in the holoenzyme.

Fatty acid synthesis in bacteria and plants is catalysed by a multi-enzyme fatty acid synthetase complex (FAS II) which consists of separate monofunctional polypeptides. Here we present a comparative molecular genetic and biochemical study of the enoyl-ACP reductase FAS components of plant and bacterial origin. The putative bacterial enoyl-ACP reductase gene (envM) was identified on the basis of amino acid sequence similarities with the recently cloned plant enoyl-ACP reductase. Subsequently, it was unambiguously demonstrated by overexpression studies that the envM gene encodes the bacterial enoyl-ACP reductase. An anti-bacterial agent called diazaborine was shown to be a specific inhibitor of the bacterial enoyl-ACP reductase, whereas the plant enzyme was insensitive to this synthetic antibiotic. The close functional relationship between the plant and bacterial enoyl-ACP reductases was inferred from genetic complementation of an envM mutant of Escherichia coli. Ultimately, envM gene-replacement studies, facilitated by the use of diazaborine, demonstrated for the first time that a single component of the plant FAS system can functionally replace its counterpart within the bacterial multienzyme complex. Finally, lipid analysis of recombinant E. coli strains with the hybrid FAS system unexpectedly revealed that enoyl-ACP reductase catalyses a rate-limiting step in the elongation of unsaturated fatty acids.