Structural basis for active-site probes targeting Staphylococcus aureus serine hydrolase virulence factors

is a major cause of infection in the community and in hospitals. Serine hydrolases play key roles in bacterial homeostasis, in particular biofilms. Activity-based profiling has previously identified a family of serine hydrolases, designated fluorophosphonate-binding hydrolases (Fphs), which contribute to virulence of in the biofilm niche. Here we report structures of the putative tributyrin esterase FphF, alone and covalently bound by a substrate analog, and small molecule inhibitors that occupy the hydrophobic substrate-binding pocket. We show that FphF has promiscuous esterase activity. Building from this, we extended our analysis to the wider Fph protein family using homology modeling and docking tools. We predict that other Fph enzymes, including FphB which was linked directly to virulence, may be more specific than FphF. This study provides insight into Fph function and a template for designing new imaging agents, diagnostic probes, and inhibitors to treat infections.