Epoxide hydrolase from Agrobacterium radiobacter

Rink R, M Eennema, M Smids, U Dehmel, DB Janssen (1997) Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter ADI. J Biol Chem 272 14650-14657. 

Rui L, 1 Cao, W Chen, KF Reardon, TK Wood (2004) Active site engineering of the epoxide hydrolase from Agrobacterium radiobacter ADI to enhance aerobic mineralization of cw-l,2-dichloroethylene in cells expressing an evolved toluene ort/io-monooxygenase. J Biol Chem 279 46810-46817. 

The first X-ray structure of an epoxide hydrolase (from Agrobacterium radiobacter ADI) has been reported recently (Fig. 11.2-4) [70f The nearly globular protein consists of a core-domain with typical features of a/(3-hydrolase fold enzymes and a so-called "cap-domain , which is located on top of the core domain. 

Interestingly, the bacterial epoxide hydrolase from Agrobacterium radiobacter ADI seems to hydrolyze para-substituted styrene oxides with opposite enantiopreference when compared to EHs from fungi or yeast[n8]. Although initial selectivities were... 

EchA epoxide hydrolase from Agrobacterium radiobacter AD1 Gly glycine... 

DESs have also proven to be suitable as cosolvent for enzymatic reactions in aqueous solution. Gorke et al. [8] have reported that upon addition of 10% of the ChCl/glycerol DES, the hydrolysis of p-nitrophenyl acetate was accelerated moderately when catalyzed by several esterases a threefold increase for pig liver esterase (PLE) and Rhizopus oryzae esterase (ROE) and a 25% increase for Pseudomonas fluorescens esterase (PFE) and CALB. The presence of the same DES in aqueous solution also triggered an up to 20-fold enhancement in the rate of the epoxide hydrolase (from Agrobacterium radiobacter)-cslaiyzed hydrolysis of styrene oxide. In fact, there was a bell-shaped relationship between the reaction rate and the DES content, as shown in Figure 25.5. 

M. Nardini, I.S. Bidder, H.J. Rozeboom, K.H. Kalk, R. Rink, D.B. Janssen, B.W. Dijkstra, The X-ray structure of epoxide hydrolase from Agrobacterium radiobacter ADI - an enzyme to detoxify harmful epoxides, J. Biol. Chem. 274 (1999) 14579-14586. 

Rink, R., Lutje Spelberg, J.H., Pieters, R.J., Kingma, J., Nardini, M., Kellogg, R.M., Dijkstra, B.W. and Janssen, D.B. (1999) Mutation of tyrosine residues involved in the alkylation half reaction of epoxide hydrolase from Agrobacterium radiobacter ADI results in improved enantioselectivity./. Am. Chem. Soc., 121, 7417-7418. 

Spelberg, J.H.L., Rink, R., Archelas, A., Furstoss, R. and Janssen, D. (2002) Biocatalytic potential of the epoxide hydrolase from Agrobacterium radiobacter ADI and a mutant with enhanced enantioselectivity. Adv. Synth. Catal., 344,980-985. 

Styrene oxide-type substrates (Fig. 2) have been hydrolyzed with good enantioselectivity especially by fungal epoxide hydrolases and a recombinant epoxide hydrolase from Agrobacterium radiobacter. However, bacterial epoxide hydrolases, e.g., from No-cardia spp. and related Rhodococcus strains, were not useful for tins substrate pattern. Similarly, enzymes from yeasts and mammals showed only low to moderate selectivity (Table 2). 

Figure 25 Conversion obtained in the hydrolysis of styrene oxide catalyzed by epoxide hydrolase ADI from Agrobacterium radiobacter as a function of the volume fraction of the ChCl/glycerol DES in aqueous solution. This figure is drawn by using the data taken from Gorke et al. [8],...

In the reaction using halohydrin dehalogenase from Agrobacterium radiobacter (Fig. 10.37(c)), the R-enantiomer was converted to the corresponding epoxide, which was further converted to (S)-diol (ee 91%) by epoxide hydrolase from the same organism to prevent attack of chloride at the /3-position. 8 The unreacted (S)-dichloropropanol was obtained in enantiomerically pure form (ee >99%). 

Most EHs have a/ 3-hydrolase fold topology and consist of a core and a lid domain [65,66]. The lid domain is mainly a-helical and contains two tyrosine residues that point toward the catalytic triad and cover the core domain. Both tyrosine residues are involved in substrate binding, Uansition-state stabilization, and activation of the epoxide by protonation. The catalytic center is composed of two aspartate and one histidine residue. The first crystal structure of an epoxide hydrolase was solved for the enzyme from Agrobacterium radiobacter ADI (EchA) [67]. The reaction mechanism of EHs is depiaed in Scheme 9.9. First, a nucleophilic attack of the aspartic residue on the epoxide ring of the substrate 31 takes place and a covalently bound ester 32 is formed. This intermediate is subsequently hydrolyzed by a so-called charge relay system (general base catalysis) and the diol 33 is released from the active site. Key reaction parmers are a histidine residue and a water molecule. It is worth mentioning that a limonene epoxide hydrolase discovered by Arand et al. displayed a different crystal structure and catalytic cycle that is discussed elsewhere [68]. 

Gao, L., Lee, J., Chen, W. and Wood, T.K. (2006) Enantioconvergent production of (R)-l-phenyl-l,2-ethanediol from styrene oxide by combining the Solatium tuberosum and an evolved Agrobacterium radiobacter ADI epoxide hydrolases. Biotechnol. Bioeng., 94, 522-529. 

---