Detoxification of environmental mutagens and carcinogens: structure, mechanism, and evolution of liver epoxide hydrolase

Abstract

The crystal structure of recombinant murine liver cytosolic epoxide hydrolase (EC 3.3.2.3) has been determined at 2.8-A resolution. The binding of a nanomolar affinity inhibitor confirms the active site location in the C-terminal domain; this domain is similar to that of haloalkane dehalogenase and shares the alpha/beta hydrolase fold. A structure-based mechanism is proposed that illuminates the unique chemical strategy for the activation of endogenous and man-made epoxide substrates for hydrolysis and detoxification. Surprisingly, a vestigial active site is found in the N-terminal domain similar to that of another enzyme of halocarbon metabolism, haloacid dehalogenase. Although the vestigial active site does not participate in epoxide hydrolysis, the vestigial domain plays a critical structural role by stabilizing the dimer in a distinctive domain-swapped architecture. Given the genetic and structural relationships among these enzymes of xenobiotic metabolism, a structure-based evolutionary sequence is postulated.

Figure 1

Ribbon plot of the epoxide hydrolase dimer, color-coded as follows: C-terminal catalytic domain, blue; N-terminal vestigial domain, green; and linker, red. Dotted green lines indicate the disordered Ala-20–Glu-47 and Val-64–Ser-89 segments in monomer A. The location of the active site is indicated by the bound inhibitor CPU. This figure was prepared with bobscript and Raster3D (–49).

Figure 2

Omit electron density map (contoured at 3.1σ) of the complex between sEH and the competitive inhibitor CPU (K_i = 3.1 nM). Note that the urea moiety of CPU binds with cis-(3-phenyl)propyl and trans-cyclohexyl amide linkages.

Figure 3

Proposed mechanism of sEH in the hydrolysis of trans-β-methylstyrene oxide. Nucleophilic attack of Asp-333 yields the alkylenzyme ester intermediate, which is subsequently hydrolyzed (with the assistance of general base His-523) to yield the vicinal diol product.

Figure 4

(Left) Least-squares superposition of the vestigial active site of sEH with the active site of haloacid dehalogenase. (Right) Least-squares superposition of the active site of sEH with that of haloalkane dehalogenase.

Figure 5

Proposed structure-based evolutionary pathway of xenobiotic catabolism. Haloalkane dehalogenase (blue) and haloacid dehalogenase (green) ancestors underwent an early gene fusion event to yield a primitive monomeric protein adopting a postulated closed conformation. Subsequent equilibration with an open conformation and dimerization through domain-swapping is facilitated by a flexible linker (i.e., hinge loop; red); stabilization of the modern-day, domain-swapped sEH dimer is achieved through subsequent shortening and/or amino acid substitutions that rigidify the 16-residue linker, which in sEH contains 5 proline residues. This figure was prepared with bobscript and Raster3D (–49).