Identification and analysis of the acyl carrier protein (ACP) docking site on beta-ketoacyl-ACP synthase III.
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Zhang YM, Rao MS, Heath RJ, Price AC, Olson AJ, Rock CO, White SW
Identification and analysis of the acyl carrier protein (ACP) docking site on beta-ketoacyl-ACP synthase III.
J Biol Chem. 2001 Mar 16;276(11):8231-8. Epub 2000 Nov 14.
- PubMed ID
- 11078736 [ View in PubMed]
- Abstract
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 ACP-protein interactions using a computational analysis to dock the NMR structure of ACP with the crystal structure of beta-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 FabH-ACP 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.