Primary Structure and Catalytic Mechanism of the Epoxide Hydrolase from Agrobacterium radiobacter AD1

摘要: The epoxide hydrolase gene fromAgrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of polymerase chain reaction with two degenerate primers based N-terminal and C-terminal sequences enzyme. coded for protein 294 amino acids molecular mass 34 kDa. An identical from chromosomal DNA closely related strain A. CFZ11. recombinant expressed up 40% total cellular content inEscherichia coli BL21(DE3) purified enzyme had akcat 21 s−1 epichlorohydrin. Amino acid sequence similarity eukaryotic hydrolases, haloalkane dehalogenase Xanthobacter autotrophicus GJ10, bromoperoxidase A2 Streptomyces aureofaciens indicated it belonged α/β-hydrolase fold family. This conclusion supported secondary structure predictions analysis circular dichroism spectroscopy. catalytic triad residues are proposed be Asp107, His275, Asp246. Replacement these Ala/Glu, Arg/Gln, Ala, respectively, resulted in dramatic loss activity mechanism proceeds via covalently bound ester intermediate, shown single turnover experiments His275 → Arg mutant which intermediate could trapped.