Limonene-1,2-epoxide hydrolase

Van der Werf Ml, KM Overkamp, JAM de Bont (1998) Limonene-1,2-epoxide hydrolase imm Rhodococcus erythropolis DCL14 belongs to a novel class of epoxide hydrolases. J Bacteriol 180 5052-5057. 

Very recently, the purification and characterisation of an epoxide hydrolase, catalysing the conversion of limonene-1,2-epoxide to limonene-1,2-diol has been described [90]. The enzyme was isolated from Rhodococcus erythropolis DCL14 and is induced when the microorganism is grown on monoterpenes. The authors found evidence that the enzyme, limonene-1,2-epoxide hydrolase is the first member of a new class (the third class) of epoxide hydrolases [91]. 

Borderline-SN2-Type Mechanism. Some enzymes, such as limonene-1,2-epoxide hydrolase, have been shown to operate via a single-step push-pull mechanism [573]. General acid catalysis by a protonated aspartic acid weakens the oxirane to facilitate a simultaneous nucleophilic attack of hydroxyl ion, which is provided by deprotonation of H2O via an aspartate anion. Due to the borderline-SN2-character of this mechanism, the nucleophile preferentially attacks the higher substituted carbon atom bearing the more stabilized 5 -charge. After liberation of the glycol, proton-transfer between both Asp-residues closes the cycle. 

Computational approaches to evaluate different mechanistic proposals for an enzyme have made great strides in the past 10 years. The chapter by Hopmann and Himo describe one such approach and its application to three different enzymatic reactions involving the transformation of an epoxide. The procedures and parameters to make a model of the active site are presented first and are followed by discussions of limonene epoxide hydrolase, soluble epoxide hydrolases, and haloalcohol dehalogenase. The results generally support the currently accepted mechanism for each enzyme but provide new insights into their regioselectivities. 

In this chapter, we will provide an overview of the employed methodology. To illustrate the various aspects of the methodology and to give the reader a feeling about the state of the art of the field, three very recent applications will be discussed in detail. All three enzymes are concerned with epoxide-transforming reactions, namely limonene epoxide hydrolase (LEH), soluble epoxide hydrolase (sEH), " and haloalcohol dehalo-genase C (HheC). First, however, a brief presentation of DFT and its accuracy will be given. 

LEH limonene epoxide hydrolase from Rhodococcus erythropolis DCL14 Leu leucine... 

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]. 

Zheng, H. and Reetz, M.T. (2010) Manipulating the stereoselectivity of limonene epoxide hydrolase by directed evolution based on iterative saturation mutagenesis. /. Am. Chem. Soc., 132, 15744 15751. 

Barbirato F, Verdoes J, de Bont J, van der Werf M (1998) The Rhodococcus erythropolis DCL14 limonene-l,2-epoxide hydrolase gene encodes an enzyme belonging to a novel class of epoxide hydrolases. FEES Lett 438 293-296... 

An application of microbial epoxide hydrolases for the synthesis of a-bisabolol, one of the stereoisomers (out of four) of interest for the cosmetic industry, is illustrated in Scheme 17. This approach was based on the diastereoselective hydrolysis of a mixture of oxirane diastereoisomers obtained by chemical synthesis from (/ )- or (5-limonene [130]. Thus, starting from (S)-limonene, the biohydrolysis of the mixture of (4S,8i S) epoxides... 

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