PRIMARY STRUCTURE AND CATALYTIC MECHANISM OF THE EPOXIDE HYDROLASE FROM AGROBACTERIUM-RADIOBACTER AD1

The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacte rium that is able to grow on epichlorohydrin as the sole carbon source , was cloned by means of the polymerase chain reaction with two degene rate primers based on the N-terminal and C-terminal sequences of the e nzyme, The epoxide hydrolase gene coded for a protein of 294 amino aci ds with a molecular mass of 34 kDa, An identical epoxide hydrolase gen e was cloned from chromosomal DNA of the closely related strain A, rad iobacter 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 epichlorohydri n, Amino acid sequence similarity of the epoxide hydrolase with eukary otic epoxide hydrolases, haloalkane dehalogenase from Xanthobacter aut otrophicus GJ10, and bromoperoxidase A2 from Streptomyces aureofaciens indicated that it belonged to the alpha/beta-hydrolase fold family, T his conclusion was supported by secondary structure predictions and an alysis 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 Al a/Glu, Arg/Gln, and Ala, respectively, resulted in a dramatic loss of activity for epichlorohydrin, The reaction mechanism of epoxide hydrol ase proceeds via a covalently bound ester intermediate, as was shown b y single turnover experiments with the His(275) --> Arg mutant of epox ide hydrolase in which the ester intermediate could be trapped.