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Pseudomonas aeruginosa Cif defines a distinct class of α/β epoxide hydrolases utilizing a His/Tyr ring-opening pair

Christopher D Bahl1, Dean R Madden

  • 1Dept. of Biochemistry, Dartmouth Medical School, 7200 Vail Building Hanover, NH 03755, USA. drm0001@dartmouth.edu

Protein and Peptide Letters
|September 22, 2011
PubMed

Insights

Pseudomonas aeruginosa virulence factor Cif, an epoxide hydrolase, requires a specific histidine for function. Mutational analysis revealed that substituting tyrosine inactivates the enzyme, highlighting unique structural requirements for Cif-like hydrolases.

Area of Science:

  • Microbiology
  • Structural Biology
  • Biochemistry

Background:

  • Pseudomonas aeruginosa is an opportunistic pathogen.
  • Cif is an epoxide hydrolase (EH) virulence factor secreted by P. aeruginosa.
  • Cif affects cystic fibrosis transmembrane conductance regulator localization.

Purpose of the Study:

  • To investigate the functional role of active site residues in Cif.
  • To determine the structural and functional consequences of substituting the catalytic tyrosine with histidine.
  • To identify potential Cif-like epoxide hydrolases in other pathogens.

Main Methods:

  • Site-directed mutagenesis to create Cif-H177Y mutant.
  • Structural analysis of the wild-type and mutant enzymes.
  • Bioinformatic analysis of pathogen genomes.

Main Results:

  • The Cif-H177Y mutant was functionally inactive.
  • Structural analysis showed that while both histidine and tyrosine fit the active site, the enzyme's conformation necessitates histidine.
  • Sequence and structure criteria identified a distinct subfamily of Cif-like EHs.

Conclusions:

  • The active site histidine is essential for Cif epoxide hydrolase activity.
  • Cif possesses unique structural features distinguishing it from canonical EHs.
  • A conserved His/Tyr pair characterizes a novel subfamily of Cif-like epoxide hydrolases.

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