Rink_1999_J.Am.Chem.Soc_121_7417

Enantiomerically pure epoxides (oxiranes) are uniquely suited building blocks for synthetic purposes. Such epoxides are often prepared by means of remarkably effective synthetic catalysts. There are, however, few enzymatic routes in the repertoire. We have studied the enzyme mediated kinetic resolution of readily available racemic epoxides by selective hydrolysis of one enan- tiomer to the 1,2-diol, a process for which a synthetic catalyst has recently also been developed. Epoxide hydrolases that perform this conversion have been found in various organisms. An attractive enzyme is the recombinant epoxide hydrolase from Agrobacterium radiobacter AD1 that can be produced in large amounts and which has good potential for the kinetic resolution of styrene oxides. Here we report novel aspects of the catalytic mechanism of the enzyme and a mechanism-based approach that has led to the first site-specific mutant of an epoxide hydrolase that has improved characteristics in kinetic resolutions. The epoxide hydrolase from A. radiobacter AD1 belongs to the alpha/beta-hydrolase fold family and contains a catalytic triad in the active site. The catalytic mechanism involves two discrete chemical steps. The first is an SN2 nucleophilic attack by an Asp107 carboxylate oxygen on the least-hindered carbon atom of the epoxide, resulting in a covalent ester intermediate. In the second step, the ester intermediate is hydrolyzed by a water molecule that is activated by the Asp246-His275 pair. The chemical opening of an epoxide is facilitated by an acidic functional group that interacts with the ring oxygen. Such an activation likely also takes place in epoxide hydrolases. Earlier speculations were made that the proton donor could be a lysine residue, but evidence in support of this is scant.8,9c, We observed from crystallographic data for epoxide hydrolase from A. radio- bacter AD1 that Tyr152 and Tyr215 are positioned close to the nucleophilic Asp107 in a manner such that their phenolic hydroxyl groups could be proton donor. No backbone amides or other acid groups are present that can serve as oxyanion hole or as proton donor during ring opening. A mechanistical role for Tyr215 was supported by a sequence alignment of known epoxide hydrolase sequences, which revealed that this tyrosine residue is absolutely conserved in the C-terminal part of the cap domain. This is remarkable considering that the overall similarity between various epoxide hydrolase sequences is often less than 20%, and indicates an important role for this residue. The tyrosine residue is conserved within a short stretch of sequence that is different for soluble and microsomal epoxide hydrolases, namely N-W/Y-Y-R and R-F/Y-Y-K, respectively. Sequence alignments were par- ticulary poor in the N-terminal part of the cap domain where Tyr152 is located, and only alignments done by hand indicated that a second tyrosine might be present in the other soluble epoxide hydrolases. To investigate the role of Tyr215 and Tyr152, we constructed mutant enzymes in which the tyrosine was replaced by a phenylalanine and the resulting mutant enzymes were expressed and purified to homogeneity.