Primary Structure and Catalytic Mechanism of the Epoxide Hydrolase from Agrobacterium radiobacterAD1

Abstract

The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme. The epoxide hydrolase gene coded for a protein of 294 amino acids with a molecular mass of 34 kDa. An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A. radiobacter CFZ11. The recombinant epoxide hydrolase was expressed up to 40% of the total cellular protein content in Escherichia coli BL21(DE3) and the purified enzyme had a k cat of 21 s ؊1 with epichlorohydrin. Amino acid sequence similarity of the epoxide hydrolase with eukaryotic epoxide hydrolases, haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, and bromoperoxidase A2 from Streptomyces aureofaciens indicated that it belonged to the ␣/␤-hydrolase fold family. This conclusion was supported by secondary structure predictions and analysis of the secondary structure with circular dichroism spectroscopy. The catalytic triad residues of epoxide hydrolase are proposed to be Asp 107 , His 275, and Asp 246 . Replacement of these residues to Ala/Glu, Arg/Gln, and Ala, respectively, resulted in a dramatic loss of activity for epichlorohydrin. The reaction mechanism of epoxide hydrolase proceeds via a covalently bound ester intermediate, as was shown by single turnover experiments with the His 275 3 Arg mutant of epoxide hydrolase in which the ester intermediate could be trapped.

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

“…Large quantities of pure epoxide hydrolase were obtained via a two-step purification procedure as described before (2). Wild-type and mutant enzyme were expressed in soluble form at a level of up to 50% of the total cellular protein content in Escherichia coli BL21(DE3).…”

“…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.…”

“…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. The catalytic triad residues Asp107, His275, and Asp246, that were also identified by mutagenesis studies (2), are positioned on small loops excursing from the -sheet. The active site is localized between the main domain and the cap domain ( Figure 1).…”

“…These enzymes have been found in many organisms, including mammals, plants, insects, fungi, and bacteria (1)(2)(3)(4). Most epoxide hydrolases play an important role in the detoxification of xenobiotic compounds, which are often degraded via reactive epoxides (5).…”

N-Acetylanthranilate Amidase fromArthrobacter nitroguajacolicusRü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

“…However, reports on protein variants of C-C hydrolase MhpC (40) and Agrobacterium radiobacter epoxide hydrolase (59) show that the observation of minor residual activity after replacement of the triad's histidine is not unprecedented. Such residual activity may be due to specific base catalysis by solvent OH Ϫ (40).…”