Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1

• Published in: The Journal of biological chemistry 1997-06, Vol.272 (23), p.14650-14657
• Main Authors: Rink, R, Fennema, M, Smids, M, Dehmel, U, Janssen, D.B
• Format: Article
• Language: English
• Subjects: AGROBACTERIUM RADIOBACTER; Amino Acid Sequence; AMINO ACID SEQUENCES; Base Sequence; BINDING SITES; Catalysis; CHEMICAL COMPOSITION; CHEMICAL STRUCTURE; Chromosomes, Bacterial; Circular Dichroism; CLONACION; CLONAGE; CLONING; Cloning, Molecular; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; DNA Primers; ENZYME REACTION MECHANISM; Epoxide Hydrolases - chemistry; Epoxide Hydrolases - isolation & purification; Epoxide Hydrolases - metabolism; Escherichia coli; ESTRUCTURA QUIMICA; GENBANK/Y12804; GENE; GENES; HIDROLASAS; HYDROLASE; HYDROLASES; MOLECULAR SEQUENCE DATA; MUTACION; Mutagenesis, Site-Directed; MUTATION; NUCLEOTIDE SEQUENCE; Point Mutation; Polymerase Chain Reaction; PROTEIN STRUCTURE; PROTEINAS RECOMBINANTES; PROTEINE RECOMBINANTE; RECOMBINANT PROTEINS; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Rhizobium - enzymology; Rhizobium - genetics; SECUENCIA NUCLEOTIDICA; Sequence Alignment; Sequence Homology, Amino Acid; SEQUENCE NUCLEOTIDIQUE; Streptomyces aureofaciens; STRUCTURE CHIMIQUE; Substrate Specificity; TARGETED MUTAGENESIS; Xanthobacter autotrophicus
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.23.14650

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 α/β-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 → Arg mutant of epoxide hydrolase in which the ester intermediate could be trapped.