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

Abstract

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.

Keywords: Pseudomonas aeruginosa; X-ray crystallography; enzyme stereospecificity; epoxide hydrolase; epoxy-fatty acids; hydroxyalkyl-enzyme intermediate; structure-function relationships; virulence factor.

Figure 1. Comparison of Cif’s interactions with EBH and EpH

A. View of the active-site residues of the aligned structures, showing the overlay of the Cif_E153Q intermediates formed with EpH (yellow carbons) and EBH (blue carbons). Inset indicates a descriptive coordinate system used throughout this manuscript. B. View after 90° rotation of the structures, with the proto-diol bonds shadowed. C. Superposition and chemical diagrams of the proposed bound epoxides as inferred from these crystal structures. The black arrows indicate the carbon attacked by Asp129. D. Specific activity of rac-EpH and rac-EBH at a 2 mM concentration. Mean ± S.D.; **, p < 0.01. See also Figure S1.

Figure 2. Hydrolysis and trapped intermediates of EpH and EpO

A. Specific activity of Cif for EpH enantiomers. B. Overlay of intermediates in crystal structures of rac-EpH (grey carbons) and S-EpH (yellow carbons). C. Specific activity of Cif for EpO enantiomers. D,E. Intermediates in the crystal structures of S-EpO (yellow carbons) and R-EpO (blue carbons), respectively. F. Adduct found in the crystal structure of rac-EpO (yellow carbons). G. Chemical diagrams of the proposed carbons attacked based on the intermediates from the structures. The black arrows indicate the carbon attacked by Asp129. Mean ± S.D.; *, p < 0.05; **, p < 0.01; ***, p < 0.001. See also Table S1 and Figures S3 and S4.

Figure 3. Hydrolysis and trapped intermediates of SOx

A. Specific activity of Cif for SOx enantiomers. B, C. View of intermediates in the crystal structures with S-SOx (yellow carbons) and R-SOx (blue carbons), respectively. D. View of intermediates in the crystal structures with rac-SOx. E. Chemical diagrams of the proposed carbons attacked by Cif based on the crystal structures. The black arrows indicate the carbon attacked by Asp129. Mean ± S.D.; *, p < 0.05; **, p < 0.01; ***, p < 0.001. See also Table S1 and Figures S3, S4, and S5.

Figure 4. Comparisons of S and R structures of substrates EpH, EpO, and SOx

A. Overlay of the S-EpO (yellow carbons) and the R-EpO (blue carbons) adduct structures. Shading highlights the direction of the shift of Asp129. B. Same as A, but with SOx. Angle symbols highlight geometric changes. C. Overlay of adduct S-substrate structures, in which the van der Waals surface of the atoms enclosing the active-site pockets with S adducts are shown in yellow mesh. D. Overlay of adduct R-substrate structures, in which the van der Waals surface of the atoms enclosing the active-site pockets with R adducts are shown in blue mesh and yellow line indicates the active site containing the S enantiomers. Black lines in C and D highlight the clashing wall of the S substrates and the pocket and the black bars indicate the point of closest contact for S-SOx. See also Figure S1.

Figure 5. Hydrolysis and trapped structure of ECH

A. Hydrolysis of ECH and VCH at a 2 mM concentration. B, C. The two ECH hydroxyalkyl-enzyme intermediates (carbons in purple). The carbon of attack determines the intermediate trapped. D. View of both intermediates in the active site, in which the van der Waals surface of the active site is shown in purple mesh. The double-headed arrow indicates the axis of rotation that allows for either carbon to be attacked. Mean ± S.D; *, p < 0.05; ***, p < 0.001. See also Table S1 and Figures S3, S4, and S6.

Figure 6. Residues shifted for accommodation of cSO and 14,15-EET

A. Schematic representations of cSO (grey bonds), R,R-tSO (blue carbons) and S,S-tSO (yellow carbons). B–E. Slices through Chain A (B–C, E) or Chain B (D) of the Cif_E153Q-cSO active site. The residues and van der Waals surfaces are shown in red for the atoms lining the active site that shift to accommodate cSO. All others protein residues and surfaces are shown in dark grey, with oxygen atoms in red. B. In the 60% of Chain A containing the cSO intermediate (grey carbons), residues Leu200 and Phe203 (red bonds) shift to accommodate the intermediate, which opens the active site to solvent. C. Top view of the active site containing the cSO intermediate. D. In Chain B, the active site is not open. The grey outline illustrates the position that would be occupied by cSO. E. Top view of the active site in C, in which cSO is replaced with the R,R-tSO (blue outline) and S,S-tSO (yellow outline) enantiomers modeled as described in the text. F. View of tunnel through Cif generated by the presence of the 14R,15S-EET intermediate (grey carbons), with colors as described for B–E. G. Three residues shift to accommodate the α tail at the right-hand side of panel F. Residues from Cif_E153Q without substrate are shown with dark grey carbons, and residues from Cif_E153Q-14,15-EET with red carbons. See also Table S1 and Figures S1, S2, S3, and S4.

Figure 7. Hydrolysis and structures of EpFAs

A. Mixed substrate hydrolysis assay. Bar colors for Cif correspond to crystal structures listed below. B. Hydrolysis of the EDP series at a 1 mM concentration. C. Hydrolysis of the EEQ series at a 1 mM concentration. D. Aligned structures of hydroxyalkyl-enzyme intermediates of 16,17-EDP (carbons and mesh surface in cyan) and 14,15-EEQ (carbons and mesh in pink). E. Aligned structures of hydroxyalkyl-enzyme intermediates of 19,20-EDP (carbons and mesh in teal) and 17,18-EEQ (carbons and mesh in purple), with Cif_E153Q without substrate (carbons and mesh in black). For D and E, the proposed trapped epoxides as inferred from the crystal structures are depicted as chemical diagrams, with the arrows indicating the carbon attacked by Asp129. F. Assay of enantiomeric excess for rac-cis-19,20-EDP. G. Examples of residue shifts near the -COOH of the EpFA intermediates, with colors as listed above. H. Residue changes between the EpFA trapped structures and the Cif_E153Q unoccupied structure occur primarily within the cap domain, as mapped onto a Cif monomer. The cap domain residues are shown in grey (no shift), yellow (loop shift in Cif_E153Q unoccupied protein), red (main chain shifts of more than 0.5 Å or side chains with conformational differences), and blue (catalytic residues). Mean ± S.D; *, p < 0.05; ***, p < 0.001. See also Table S2 and Figures S3, S5, and S7.