Identification and analysis of the acyl carrier protein (ACP) docking siteon beta-ketoacyl-ACP synthase III

The molecular details that govern the specific interactions between acyl ca rrier protein (ACP) and the enzymes of fatty acid biosynthesis are unknown. We investigated the mechanism of ACP protein interactions using a computat ional analysis to dock the NMR structure of ACP with the crystal structure of beta -ketoacyl-ACP synthase III (FabH) and experimentally tested the mod el by the biochemical analysis of FabH mutants. The activities of the mutan ts 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-independen t assay. ACP docked to a positively charged/hydrophobic patch adjacent to t he active site tunnel on FabH, which included a conserved arginine (Arg-249 ) that was required for ACP docking. Kinetic analysis and direct binding st udies between FabH and ACP confirmed the identification of Arg-249 as criti cal for FabH ACP interaction. Our experiments reveal the significance of th e positively charged/hydrophobic patch located adjacent to the active site cavities of the fatty acid biosynthesis enzymes and the high degree of sequ ence conservation in helix II of ACP across species.