Evolution of tunnels in alpha/beta-hydrolase fold proteins - what can we learn from studying epoxide hydrolases?

The evolutionary variability of a protein's residues is highly dependent on protein region and protein function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues. Active site residues are considered to be conserved as they ensure an enzyme's activity and selectivity. The abovementioned rules apply also to α/β-hydrolase fold proteins - an example of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We hypothesised two scenarios: (1) tunnels are lined by mostly variable residues, allowing adaptation to the evolutionary pressures of a changeable environment; or (2) tunnels are lined by mostly conserved amino acids, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. We also wanted to check if evolutionary analysis can help distinguish functional and non-functional tunnels. Soluble epoxide hydrolases (sEHs) represent a good case study for the analysis of the evolution of tunnels in an α/β-hydrolase fold family due to their size and architecture. Here, we propose methods for the comparison of tunnels detected in both crystal structures and molecular dynamics simulations, as well as the assignment of tunnel functionality, and we identify critical steps for careful tunnel inspection. We also compare the entropy values of the tunnel-lining residues and system-specific compartments in seven selected sEHs from different clades. We present three different cases of entropy distribution among tunnel-lining residues. As a result, we propose a 'perforation' model for tunnel evolution via the merging of internal cavities or surface perforations. We also report an approach for the identification of highly variable tunnel-lining residues as potential targets to be used for the fine-tuning of selected enzymes.

Relative Importance of Soluble and Microsomal Epoxide Hydrolases for the Hydrolysis of Epoxy-Fatty Acids in Human Tissues

Epoxy-fatty acids (EpFAs) are endogenous lipid mediators that have a large breadth of biological activities, including the regulation of blood pressure, inflammation, angiogenesis, and pain perception. For the past 20 years, soluble epoxide hydrolase (sEH) has been recognized as the primary enzyme for degrading EpFAs in vivo. The sEH converts EpFAs to the generally less biologically active 1,2-diols, which are quickly eliminated from the body. Thus, inhibitors of sEH are being developed as potential drug therapeutics for various diseases including neuropathic pain. Recent findings suggest that other epoxide hydrolases (EHs) such as microsomal epoxide hydrolase (mEH) and epoxide hydrolase-3 (EH3) can contribute significantly to the in vivo metabolism of EpFAs. In this study, we used two complementary approaches to probe the relative importance of sEH, mEH, and EH3 in 15 human tissue extracts: hydrolysis of 14,15-EET and 13,14-EDP using selective inhibitors and protein quantification. The sEH hydrolyzed the majority of EpFAs in all of the tissues investigated, mEH hydrolyzed a significant portion of EpFAs in several tissues, whereas no significant role in EpFAs metabolism was observed for EH3. Our findings indicate that residual mEH activity could limit the therapeutic efficacy of sEH inhibition in certain organs.

Mycobacterial Epoxide Hydrolase EphD Is Inhibited by Urea and Thiourea Derivatives

The genome of the human intracellular pathogen Mycobacterium tuberculosis encodes an unusually large number of epoxide hydrolases, which are thought to be involved in lipid metabolism and detoxification reactions needed to endure the hostile environment of host macrophages. These enzymes therefore represent suitable targets for compounds such as urea derivatives, which are known inhibitors of soluble epoxide hydrolases. In this work, we studied in vitro the effect of the thiourea drug isoxyl on six epoxide hydrolases of M. tuberculosis using a fatty acid substrate. We show that one of the proteins inhibited by isoxyl is EphD, an enzyme involved in the metabolism of mycolic acids, key components of the mycobacterial cell wall. By analyzing mycolic acid profiles, we demonstrate the inhibition of EphD epoxide hydrolase activity by isoxyl and two other urea-based inhibitors, thiacetazone and AU1235, inside the mycobacterial cell.

Biochemical and Structural Characterization of Two Cif-Like Epoxide Hydrolases from Burkholderia cenocepacia

Epoxide hydrolases catalyze the conversion of epoxides to vicinal diols in a range of cellular processes such as signaling, detoxification, and virulence. These enzymes typically utilize a pair of tyrosine residues to orient the substrate epoxide ring in the active site and stabilize the hydrolysis intermediate. A new subclass of epoxide hydrolases that utilize a histidine in place of one of the tyrosines was established with the discovery of the CFTR Inhibitory Factor (Cif) from Pseudomonas aeruginosa. Although the presence of such Cif-like epoxide hydrolases was predicted in other opportunistic pathogens based on sequence analyses, only Cif and its homologue aCif from Acinetobacter nosocomialis have been characterized. Here we report the biochemical and structural characteristics of Cfl1 and Cfl2, two Cif-like epoxide hydrolases from Burkholderia cenocepacia. Cfl1 is able to hydrolyze xenobiotic as well as biological epoxides that might be encountered in the environment or during infection. In contrast, Cfl2 shows very low activity against a diverse set of epoxides. The crystal structures of the two proteins reveal quaternary structures that build on the well-known dimeric assembly of the α/β hydrolase domain, but broaden our understanding of the structural diversity encoded in novel oligomer interfaces. Analysis of the interfaces reveals both similarities and key differences in sequence conservation between the two assemblies, and between the canonical dimer and the novel oligomer interfaces of each assembly. Finally, we discuss the effects of these higher-order assemblies on the intra-monomer flexibility of Cfl1 and Cfl2 and their possible roles in regulating enzymatic activity.