Crystal structure of the cystic fibrosis transmembrane conductance regulator inhibitory factor cif reveals novel active-site features of an epoxide hydrolase virulence factor

Christopher D. Bahl, Christophe Morisseau, Jennifer M. Bomberger, Bruce A. Stanton, Bruce D. Hammock, George A. O'Toole, Dean R. Madden

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other α/β hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-Å resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of α/β hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.

Original languageEnglish (US)
Pages (from-to)1785-1795
Number of pages11
JournalJournal of Bacteriology
Volume192
Issue number7
DOIs
StatePublished - Apr 2010

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Epoxide Hydrolases
Cystic Fibrosis Transmembrane Conductance Regulator
Virulence Factors
Catalytic Domain
haloalkane dehalogenase
haloacetate dehalogenase
Epoxy Compounds
Hydrolases
Hydrolysis
X Ray Crystallography
Substrate Specificity
Pseudomonas aeruginosa
Sequence Analysis
Epithelial Cells
Mutation
Membranes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Crystal structure of the cystic fibrosis transmembrane conductance regulator inhibitory factor cif reveals novel active-site features of an epoxide hydrolase virulence factor. / Bahl, Christopher D.; Morisseau, Christophe; Bomberger, Jennifer M.; Stanton, Bruce A.; Hammock, Bruce D.; O'Toole, George A.; Madden, Dean R.

In: Journal of Bacteriology, Vol. 192, No. 7, 04.2010, p. 1785-1795.

Research output: Contribution to journalArticle

Bahl, Christopher D. ; Morisseau, Christophe ; Bomberger, Jennifer M. ; Stanton, Bruce A. ; Hammock, Bruce D. ; O'Toole, George A. ; Madden, Dean R. / Crystal structure of the cystic fibrosis transmembrane conductance regulator inhibitory factor cif reveals novel active-site features of an epoxide hydrolase virulence factor. In: Journal of Bacteriology. 2010 ; Vol. 192, No. 7. pp. 1785-1795.
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abstract = "Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other α/β hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-{\AA} resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of α/β hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.",
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