Epoxide hydrolases sources

It was recently reported that. >97% of BaP 4,5-epoxide metabolically formed from the metabolism of BaP in a reconstituted enzyme system containing purified cytochrome P-450c (P-448) is the 4S,5R enantiomer (24). The epoxide was determined by formation, separation and quantification of the diastereomeric trans-addition products of glutathione. Recently a BaP 4,5-epoxide was isolated from a metabolite mixture obtained from the metabolism of BaP by liver microsomes from 3-methylcholanthrene-treated Sprague-Dawley rats in the presence of the epoxide hydrolase inhibitor 3,3,3-trichloropropylene oxide, and was found to contain a 4S,5R/4R,5S enantiomer ratio of 94 6 (Chiu et. al., unpublished results). However, the content of the 4S,5R enantiomer was <60% when liver microsomes from untreated and phenobarbital-treated rats were used as the enzyme sources. Because BaP 4R,5S-epoxide is also hydrated predominantly to 4R,5R-dihydro-... 

In eukaryotes, such as mammals and fungi, epoxide hydrolases play a key role in the metabolism of xenobiotics, in particular of aromatic systems [30,31 ]. On the other hand, in prokaryotes (e.g. bacteria) these enzymes are essential for the utilization of alkenes as carbon-source. In general, aromatics can be metabolized via two different pathways (Scheme 5) (i) dioxetane formation via dioxyge-... 

It is noteworthy that, in contrast to mammalian systems, the majority of bacterial strains exhibited sufficient activity even when the cells were grown under non-optimized conditions. Since enzyme induction is still a largely empirical task, cells were grown on standard media in the absence of inducers. Furthermore, all attempts to induce epoxide hydrolase activity in Pseudomonas aeruginosa NCIMB 9571 and Pseudomonas oleovorans ATCC 29347 by growing the cells on an alkane (decane) or alkene (1-octene) as the sole carbon source failed [27]. 

Although the use of an epoxide hydrolase was already claimed for the industrial synthesis of L- and meso-tartaric acid in 1969 [51], it was only recently that applications to asymmetric synthesis appeared in the hterature. This fact can be attributed to the limited availabihty of these biocatalysts from sources such as mammals or plants. Since the production of large amounts of crude enzyme is now feasible, preparative-scale applications are within reach of the synthetic chemist. For instance, fermentation of Nocardia EHl on a 701-scale afforded > 700 g of lyophilized cells [100]). 

An attractive alternative to the methods mentioned above is the use of cofactor-independent epoxide hydrolases, which are readily available from microbial sources in sufficient quantities. 

In spite of the considerable value of epoxide hydrolases for fine chemical synthesis, it was only recently that a detailed search for epoxide hydrolases from microbial sources was undertaken by the groups of Furstoss185, 901 and Faber123, 79, 911, bearing in mind that the use of microbial enzymes allows an (almost) unlimited supply of biocatalyst. The screening was based along the following considerations on the one hand, the catabolism of alkenes often implies the hydrolysis of an epoxide inter-... 

Trisubstituted Epoxides. To date, only a limited set of data are available on the enzymatic hydrolysis of trisubstituted epoxides (Table 11.2-7). Regardless of their steric bulkiness, however, they seem to be accepted by epoxide hydrolases from bacterial1110, 119], fungal[92, 941 and yeast[9S1 sources, as long as the access to one side of the substrate is not too severely restricted (e.g. a 2,2-dimethyl-3-alkyloxirane). Further data are required to depict a general selectivity pattern within this group of substrates. 

Over the past few years, an impressive array of epoxide hydrolases has been identified from microbial sources. Due to the fact that they can be easily employed as whole-cell preparations or crude cell-free extracts in sufficient amounts by fermentation, they are just being recognized as highly versatile biocatalysts for the preparation of enantiopure epoxides and vicinal diols. The future will certainly bring an increasing number of useful applications of these systems to the asymmetric synthesis of chiral bioactive compounds. As for all enzymes, the enantioselectivity of... 

The hydrolysis of epoxides to give 1 -diols is an area that is ripe for development. Some work has been published showing that epoxides such as cyclohexane epoxide (36) form optically active diols, in this case cyclo-hexane-(lR,2i )-diol (37). The research has concentrated on the use of enzymes present in liver microsomes, and while this elegant work has indicated what can be achieved, it is clear that rapid progress and the involvement of non-experts in this particular area must await the discovery of readily available epoxide hydrolase enzyme(s) from microbial sources. 

Although many studies have been undertaken with hepatic epoxide hydrolases, mainly aimed at the elucidation of detoxification mechanisms, it is unlikely that enzymes from these sources will be widely used as biocatalysts in preparative... 

As a result, an impressive amount of knowledge on microbial epoxide hydrolases from various sources - such as bacteria, filamentous fungi, and yeasts - has been gathered and featured in several reviews [594-598]. The data available to date indicate that the enantioselectivities of enzymes from certain microbial sources can be correlated to the substitutional pattern of various types of substrates [599] ... 

Among the sterically more demanding substrates, 2,2-disubstituted oxiranes were hydrolyzed in virtually complete enantioselectivities using enzymes from bacterial sources (E > 200), in particular Mycobacterium NCIMB 10420, Rhodococcus (NCIMB 1216, DSM 43338, IFO 3730) and closely related Nocardia spp. (Scheme 2.93) [608, 609]. All bacterial epoxide hydrolases exhibited a preference for the (S)-enantiomer. In those cases where the regioselectivity was determined, attack was found to exclusively occur at the unsubstituted oxirane carbon atom. 

Kotik, M., Stepanek, V., Maresova, H., Kyslik, P. and Archelas, A. (2009) Environmental DNA as a source of a novel epoxide hydrolase reacting with aliphatic terminal epoxides. /. Mol Catal B Enzym., 56, 288-293. 

Epoxide hydrolases have been purified from mammalian liver cells [63-66] but also from microbial sources such as Bacillus megaterium [67], Corynebacterium [45,46,68], Pseudomonas sp. [46,69], and dematiaceous fungi such as Ulocladium atrum and Zopfiella karachiensis [70]. However, some of these enzymes were only partially purified [67,68], or their enantioselectivity was not investigated [69] or was very low [45,46]. More recently, two bacterial epoxide hydrolases with high activity and high enantioselectivity were purified and characterized from Rhodococcus sp. NCIMB 11216 [71] and Nocardia TBl [72]. 

Regardless of the biological source, all epoxide hydrolases found to date do not possess a metal atom or a prosthetic group, and do not require any cofactors. Since the overexpression of a mammalian epoxide hydrolase did not lead to a highly active enzyme... 

The search for novel microbial epoxide hydrolases through title screening of various fungal and bacterial sources was mainly triggered by chance observations of unexpected side reactions in microbial transformations within two research groups at about the same time. 

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