BPS2026 – Elucidating substrate recruitment and transport mechanism in oleate hydratase

Staphylococcus aureus ( S . aureus ) is a major human pathogen responsible for a spectrum of diseases, from minor skin infections to severe conditions such as pneumonia and septic arthritis. Its ability to evade host immune defenses is mediated by multiple virulence factors, including oleate hydratase (OhyA) that converts host-derived antimicrobial unsaturated fatty acids (uFAs), typically oleic acid, into anti-inflammatory hydroxystearic acid, thereby aiding bacterial survival. Previously, we derived a membrane-bound model of OhyA based on its crystal structure using molecular dynamics (MD) simulations, which indicates that the active site would be ∼40 Å away from the membrane. Although crystal structures of OhyA reveal a hydrophobic tunnel leading to the active site, the exact mechanism of substrate recruitment from the membrane, entry into the tunnel, and subsequent transport remains unclear. To address this gap, we simulated the membrane-bound OhyA in a S. aureus native-like bilayer and captured the spontaneous elevation of a phospholipid toward the tunnel entrance, suggesting that initial substrate extraction from the membrane is facilitated by electrostatic interactions with lysine residues at the tunnel entrance. To investigate the full transport process, we replaced the elevated lipid with its natural substrate, oleate, and applied external force to guide it through the hydrophobic tunnel (∼30 Å) using steered MD (SMD). Surprisingly, the oleate anion does not enter the tunnel readily, whereas its conjugate acid, the neutral oleic acid, requires much less external force to transport through the tunnel and reach the active site. This suggests that substrate protonation might be involved within the protein environment. Our study provides molecular insights into substrate recognition and transport pathway in OhyA, offering a deeper understanding of this critical S. aureus virulence factor and laying a foundation for rational design of therapeutic strategies targeting detoxification of uFAs.