Pseudomonas aeruginosa Cif Defines a Distinct Class of α/β Epoxide Hydrolases Utilizing a His/Tyr Ring-Opening Pair

Abstract

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that secretes a multitude of virulence factors during the course of infection. Among these is Cif, an epoxide hydrolase (EH) that reduces the functional localization of the cystic fibrosis transmembrane conductance regulator in epithelial cells. In addition to being the first reported EH virulence factor, Cif possesses unique sequence deviations from canonical EH motifs. Foremost among these is the substitution of a histidine for the second epoxide ring-opening tyrosine in the active site. To test the functional equivalence of the Tyr and His side chains at this position, we have generated the mutant Cif-H177Y. Structural analysis confirms that both the WT His and mutant Tyr side chains can be accommodated without large-scale conformational changes. However, the Tyr mutation is functionally inactive. Based on a detailed analysis of the structure of the Tyr mutant, it appears that Cif's main-chain conformation imposes a functional requirement for a His at this position. Comparison with canonical EH structures reveals additional conformational differences, which are coupled to divergent sequence characteristics. When used to probe the genomes of other opportunistic pathogens, these sequence-structure criteria uncover candidate sequences that appear to form a distinct subfamily of Cif-like epoxide hydrolases characterized by a conserved His/Tyr ring-opening pair.

Signature Motifs Identify an Acinetobacter Cif Virulence Factor with Epoxide Hydrolase Activity

“…Most EH enzymes possess an active site buried within the interior of the protein with two outlets to the solvent. The shifted position of this helix in the Cif structure caps one opening of the active-site tunnel, converting the active-site tunnel into a pocket (29). This helix is in a nearly identical position in the aCif and Cif structures (blue and yellow helices in Fig.…”

“…Structural Analysis of aCif-aCif was identified as a putative Cif-like EH because it shares with Cif a set of active-site sequence motifs that differ from those conserved among canonical EH family members (29). To test our hypothesis directly that these motifs adopt Cif-like structures in the context of the aCif sequence, we crystallized aCif and determined its structure by x-ray diffraction analysis.…”

“…Classically, EHs enzymes have utilized a pair of Tyr residues for this task (49). In contrast, Cif uses a His/Tyr pair, a combination also used to position substrates in the fluoroacetate dehalogenases (27,29). aCif shares this altered His/Tyr motif, and the active site clearly displays His and Tyr side chains aligned in a nearly identical position and orientation as the Cif ring-opening pair (Fig.…”

“…This residue projects into the active site near the junction between the ␣/␤ hydrolase core and cap domains of Cif, whereas it is commonly located within the ␣/␤ hydrolase core domain of canonical EHs (29). aCif also possess an acidic residue forming similar hydrogen bonds and at the position equivalent to the charge-relay acid of Cif (Fig.…”

“…Subsequent analysis revealed a series of structural adaptations that enable Cif to retain EH activity despite sequence variations in these active site residues (27,29). Sequence comparisons uncovered similar motifs in the genomes of other opportunistic pathogens, and sequence-structure mapping suggested that these putative EHs are likely to adopt a conformation similar to Cif (29).…”