The X-ray Structure of Epoxide Hydrolase from Agrobacterium radiobacter AD1

Abstract

Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides. They play an essential role in the detoxification of various xenobiotics in higher organisms and in the bacterial degradation of several environmental pollutants. The first x-ray structure of one of these, from Agrobacterium radiobacter AD1, has been determined by isomorphous replacement at 2.1-Å resolution. The enzyme shows a two-domain structure with the core having the ␣/␤ hydrolase-fold topology. The catalytic residues, Asp 107 and His 275 , are located in a predominantly hydrophobic environment between the two domains. A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His 275 . Because of a crystallographic contact, the active site has become accessible for the Gln 134 side chain, which occupies a position mimicking a bound substrate. The structure suggests Tyr 152 /Tyr 215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen.Epoxide hydrolases (EC 3.3.2.3) are a group of functionally related enzymes that catalyze the cofactor-independent hydrolysis of epoxides to their corresponding diols by the addition of a water molecule. Epoxides are very reactive electrophilic compounds frequently found as intermediates in the catabolic pathway of various xenobiotics. For instance they are the carcinogens formed by bioactivation reactions catalyzed by cytochrome P450. Therefore, conversion of epoxides to less toxic, watersoluble compounds is an essential detoxification step in living cells. Consequently, epoxide hydrolases have been found in a wide variety of organisms, including mammals, invertebrates, plants, and bacteria (1).Until now most research has been focused on mammalian epoxide hydrolases (2, 3), which, together with glutathione S-transferases, are the most important enzymes to convert toxic epoxides to more polar and easily excretable compounds (4). However, much progress has recently also been made in the characterization of bacterial epoxide hydrolases (5, 6, 7). These enzymes show a significant sequence homology with those of mammalian origin. They can be easily obtained in large amounts, and they exhibit enantioselectivity with various industrially important epoxides, which makes them promising biocatalysts for the large scale preparation of enantiopure epoxides and/or their corresponding vicinal diols (8). In particular, extensive studies have been performed on the epoxide hydrolase from Agrobacterium radiobacter AD1, a Gram-negative bacterium that is able to use the environmental pollutant epichlorohydrin as its sole carbon and energy source (5,6,8). This epoxide hydrolase is a soluble monomeric globular protein of 35 kDa with a broad substrate range. Epichlorohydrin and epibromohydrin are its best substrates, and the optimum pH range for catalysis...

Tyrosine Residues Serve as Proton Donor in the Catalytic Mechanism of Epoxide Hydrolase from Agrobacterium radiobacter

“…FIGURE 1: View of the active site residues as observed in the X-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1 (9). The catalytic triad residues Asp107 and His275, the position of Gln134, and the two catalytically active tyrosine residues are shown as open sticks, and the other residues are shown in black.…”

“…The effect of pH on log(k cat /K m ) of the wild-type enzyme (b), the Y215F mutant (9), and the Y152F mutant (0) was determined with (R)-PNSO at 30°C. The solid lines are fits of the data to eq 3.…”

“…The X-ray structure of epoxide hydrolase from A. radiobacter AD1 was recently determined (9). This enabled the identification of additional residues besides the catalytic triad that may have a function in enzyme catalysis (9).…”

“…The 34 kDa enzyme is enantioselective toward styrene oxide and substituted derivatives thereof (8). The X-ray structure was recently solved, and it shows that this epoxide hydrolase has an R/ -hydrolase fold structure (9,10), as was expected from the sequence (2). The threedimensional structure consists of two domains: a main domain with a central -sheet composed of eight strands surrounded by six R-helices and a cap domain that is composed of five R-helices, which lies on top of the main domain.…”

“…According to the X-ray structure of epoxide hydrolase, two tyrosine residues are probably involved in the cleavage of the epoxide ring (9). The phenolic hydroxyl groups of the cap domain residues Tyr215 and Tyr152 are in a position to form hydrogen bonds with the oxirane oxygen of enzymebound substrate.…”