Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase

• Published in: Structure (London) 2017-05, Vol.25 (5), p.697-707.e4
• Main Authors: Hvorecny, Kelli L., Bahl, Christopher D., Kitamura, Seiya, Lee, Kin Sing Stephen, Hammock, Bruce D., Morisseau, Christophe, Madden, Dean R.
• Format: Article
• Language: English
• Subjects: Binding Sites; Crystallography, X-Ray; enzyme stereospecificity; epoxide hydrolase; Epoxide Hydrolases - chemistry; Epoxide Hydrolases - metabolism; epoxy-fatty acids; hydroxyalkyl-enzyme intermediate; Molecular Dynamics Simulation; Protein Binding; Pseudomonas aeruginosa; Pseudomonas aeruginosa - enzymology; structure-function relationships; Substrate Specificity; virulence factor; Virulence Factors - chemistry; Virulence Factors - metabolism; X-ray crystallography
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2017.03.002

Pseudomonas aeruginosa secretes an epoxide hydrolase with catalytic activity that triggers degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and perturbs other host defense networks. Targets of this CFTR inhibitory factor (Cif) are largely unknown, but include an epoxy-fatty acid. In this class of signaling molecules, chirality can be an important determinant of physiological output and potency. Here we explore the active-site chemistry of this two-step α/β-hydrolase and its implications for an emerging class of virulence enzymes. In combination with hydrolysis data, crystal structures of 15 trapped hydroxyalkyl-enzyme intermediates reveal the stereochemical basis of Cif's substrate specificity, as well as its regioisomeric and enantiomeric preferences. The structures also reveal distinct sets of conformational changes that enable the active site to expand dramatically in two directions, accommodating a surprising array of potential physiological epoxide targets. These new substrates may contribute to Cif's diverse effects in vivo, and thus to the success of P. aeruginosa and other pathogens during infection. [Display omitted] •Adducts probe the active-site geometry of an epoxide hydrolase virulence factor•Localized conformational flexibility enables Cif to hydrolyze epoxy-fatty acids•Substrate competition can modulate Cif-specific activity for targets in vivo•Cif allows P. aeruginosa to perturb important host regulatory signals Hvorecny et al. crystallized covalent intermediates of the epoxide hydrolase virulence factor Cif. The structures collectively illuminate Cif's stereospecificity and uncover a new set of fatty acid substrates. Cif can thus target key host immune signals, exacerbating the damage caused by opportunistic pathogens such as P. aeruginosa.