Seeing Is Believing: Visualizing Hydrolysis and Inhibition of an Epoxide Hydrolase Virulence Factor

Chronic obstructive pulmonary disease (COPD), the third leading cause of death in the US, results in poor mucociliary clearance and increased lung colonization by opportunistic pathogens, such as Pseudomonas aeruginosa ( Pa ). Pa drives a decrease in levels of the ion channel CFTR, which regulates the fluid balance at the air‐liquid interface of the lungs for proper mucociliary clearance. Pa acts on CFTR through the secretion of an epoxide hydrolase, CFTR Inhibitory Factor (Cif). Here we show that Pa decreases mucociliary transport in vitro . Deletion of the Cif gene from Pa prevents this decrease. Cystic fibrosis patients, a group chronically infected with Pa , harbor serum antibodies to Cif, indicating that Pa secretes Cif in situ . Further work ties Cif's hydrolase activity to the decrease of CFTR and thus to the observed mucociliary transport defect. To gain an understanding of Cif's substrate selectivity and enzyme kinetics, we employed hydrolase null mutants and small molecule inhibitors. Utilizing a combination of X‐ray crystallography and fluorogenic reporter assays, we can describe Cif's steric requirements for substrates and the cross talk occurring between the monomers of the dimer during hydrolysis and inhibition. This work provides a rare look into the enzymatic intermediates of a bacterial toxin, and produces structural insights key to the development of second‐generation inhibitors with the potential to act as a complement to antibiotic and disease therapies Funding: NIH and CFF