From stability to specificity: mechanistic insights into the functions of two acyl carrier proteins, ApeE and ApeF, in aryl polyene biosynthesis in A. baumannii

Aryl polyene (APE) biosynthesis in Acinetobacter baumannii relies on two adjacent acyl carrier proteins, ApeE and ApeF, that share a conserved fold but display significant sequence, structural, and functional asymmetry. We aimed to elucidate how the specialization of these two ACPs arises from differences in substrate-binding geometry and intrinsic stabilization of their folds. Comparative analysis reveals that ApeE exhibits greater conformational plasticity, enabling it to accommodate bulky APE intermediates, reflecting its role in flexible substrate handling. Conversely, ApeF features a shallow hydrophobic cavity and globally rigid architecture, characterized by a compact hydrophobic core and localized dynamic regions, crucial for malonyl-group transfer. Thermal and chemical denaturation experiments demonstrate that ApeF possesses a global unfolding free energy of 4.61 kcal·mol⁻¹ and a melting temperature of 72.7 °C, compared to ApeE’s 2.24 kcal·mol⁻¹ and 64.7 °C, highlighting its superior stability. NMR relaxation and hydrogen/deuterium exchange analyses indicated localized dynamics near the prosthetic group attachment site in holo-ApeF, with greater localized protection at residues I50, V72, and V73, exhibiting markedly slower exchange than in ApeE, which correlates with greater stability and rigidity. Molecular dynamics simulations corroborate that ApeF maintains structural integrity during dynamic substrate interactions, while ApeE’s flexibility facilitates substrate switching necessary for APE biosynthesis. These findings highlight the evolutionary adaptations that optimize each ACP’s function within the biosynthetic pathway, providing critical insights into the balance between stability and flexibility in substrate specificity and protein structure-function relationships. Ultimately, our study demonstrates how subtle variations within a conserved framework contribute to significant functional diversity.