H333N] was significantly more resistant to both antibiotics than FabB and had an affinity for TLM an order of magnitude less than the wild-type enzyme, illustrating that the two-histidine active site architecture is critical to protein-antibiotic interaction. These data provide a structural framework for understanding antibiotic sensitivity within this group of enzymes.

The molecular details that govern the specific interactions between acyl carrier protein (ACP) and the enzymes of fatty acid biosynthesis are unknown. We investigated the mechanism of ACP⅐protein interactions using a computational analysis to dock the NMR structure of ACP with the crystal structure of ␤-ketoacyl-ACP synthase III (FabH) and experimentally tested the model by the biochemical analysis of FabH mutants. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The ACP interaction surface was defined by mutations that compromised FabH activity in the ACP-dependent assay but had no effect in the ACP-independent assay. ACP docked to a positively charged/hydrophobic patch adjacent to the active site tunnel on FabH, which included a conserved arginine (Arg-249) that was required for ACP docking. Kinetic analysis and direct binding studies between FabH and ACP confirmed the identification of Arg-249 as critical for FabH⅐ACP interaction. Our experiments reveal the significance of the positively charged/ hydrophobic patch located adjacent to the active site cavities of the fatty acid biosynthesis enzymes and the high degree of sequence conservation in helix II of ACP across species.The 4Ј-phosphopantetheine prosthetic group is a central and universal feature in the mechanism of fatty acid biosynthesis that provides two crucial functionalities to the process: a long and flexible arm that can reach into active sites and a terminal sulfhydryl group for the attachment of acyl groups through a thioester linkage. Two types of fatty acid synthase architectures exist in nature, and the 4Ј-phosphopantetheine moiety operates quite differently in each type. The type I, or associated system, found in metazoans, consists of a single large polypeptide containing multiple active centers. In this system, the prosthetic group with its attached nascent fatty acid swings between the active sites in the multifunctional complex. This contrasts with the type II or dissociated system found in bacteria and plants in which the active centers reside in discrete protein molecules. Here, the 4Ј-phosphopantetheine moiety is covalently attached to acyl carrier protein (ACP), 1 a small protein that sequentially delivers the lipid intermediates to the active site of each enzyme in the pathway.ACP is a small, acidic and highly conserved protein with a molecular mass of 8847 Da (1). In Escherichia coli, it is encoded by the acpP gene that is located within a cluster of other fatty acid biosynthetic genes (2, 3). Biophysical (4) and solution NMR structural studies (5) show that the protein is an asymmetric monomer composed of three ␣-helices packed into a bundle with an extended and flexible loop at one end (6, 7). The fatty acid intermediates are attached to the terminal sulfhydryl of the 4Ј-phosphopantetheine prosthetic group, which in turn, is covalently attached to serine 36 via a phosphodiester linkage (8). In addition to fatty acid biosynthesis, ACP also supplies acyl groups for the synthesis ...

beta-Ketoacyl-acyl carrier protein reductase (FabG) is a key component in the type II fatty acid synthase system. The structures of Escherichia coli FabG and the FabG[Y151F] mutant in binary complexes with NADP(H) reveal that mechanistically important conformational changes accompany cofactor binding. The active site Ser-Tyr-Lys triad is repositioned into a catalytically competent constellation, and a hydrogen bonded network consisting of ribose hydroxyls, the Ser-Tyr-Lys triad, and four water molecules creates a proton wire to replenish the tyrosine proton donated during catalysis. Also, a disordered loop in FabG forms a substructure in the complex that shapes the entrance to the active site. A key observation is that the nicotinamide portion of the cofactor is disordered in the FabG[Y151F].NADP(H) complex, and Tyr151 appears to be necessary for high-affinity cofactor binding. Biochemical data confirm that FabG[Y151F] is defective in NADPH binding. Finally, structural changes consistent with the observed negative cooperativity of FabG are described.

The structure of beta-ketoacyl-[acyl carrier protein] reductase (FabG) from Escherichia coli was determined via the multiwavelength anomalous diffraction technique using a selenomethionine-labeled crystal containing 88 selenium sites in the asymmetric unit. The comparison of the E. coli FabG structure with the homologous Brassica napus FabG.NADP(+) binary complex reveals that cofactor binding causes a substantial conformational change in the protein. This conformational change puts all three active-site residues (Ser 138, Tyr 151, and Lys 155) into their active configurations and provides a structural mechanism for allosteric communication between the active sites in the homotetramer. FabG exhibits negative cooperative binding of NADPH, and this effect is enhanced by the presence of acyl carrier protein (ACP). NADPH binding also increases the affinity and decreases the maximum binding of ACP to FabG. Thus, unlike other members of the short-chain dehydrogenase/reductase superfamily, FabG undergoes a substantial conformational change upon cofactor binding that organizes the active-site triad and alters the affinity of the other substrate-binding sites in the tetrameric enzyme.