Effect of Cargo Identity on ACYL Carrier Protein Structure

Abstract

Biosynthesis of many primary and secondary metabolites depends on a carrier protein to transfer intermediates between appropriate enzymes. In type II fatty acid biosynthesis, hydrophobic substrates are sequestered inside the cavity of the acyl carrier protein (ACP), and protein-protein interactions precede chain flipping of the substrate from ACP into the active site of the enzyme. Due to the vast amount of substrate and enzyme combinations possible, it is likely that a selective, rather than stochastic, mechanism drives chain flipping. Little is understood regarding how the identity of ACP cargo is communicated to partner enzymes while sequestered inside the hydrophobic pocket. We show that subtle, even single atom, differences can have large effects on the overall protein structure. We present a method which allows stability of substrate-loaded ACP through incorporation of the substrate attachment enzyme during NMR to define the effect of substrate identity on protein structure. This insight will lead to improved understanding of carrier protein dependent pathways, which is necessary for re-engineering these pathways for desired products.