Access to enantiopure aromatic epoxides and diols using epoxide hydrolases derived from total biofilter DNA

Kotik, Michael; Štěpánek, Václav; Grulich, Michal; Kyslı́k, Pavel; Archelas, Alain

doi:10.1016/j.molcatb.2010.01.016

Cited by 56 publications

(51 citation statements)

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“…Although the substrate specificity shown by Rv2740 is fairly different from that of Re-LEH, the available information about this enzyme is quite preliminary and no real application for its synthetic exploitation has been made. Sequence-based mining of (meta)genomes for novel EHs has been limited so far to the discovery of enzymes showing an a/b-hydrolase fold [16][17][18].…”

Section: Introductionmentioning

confidence: 99%

Discovery and characterization of thermophilic limonene‐1,2‐epoxide hydrolases from hot spring metagenomic libraries

et al. 2015

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The epoxide hydrolases (EHs) represent an attractive option for the synthesis of chiral epoxides and 1,2-diols which are valuable building blocks for the synthesis of several pharmaceutical compounds. A metagenomic approach has been used to identify two new members of the atypical EH limonene-1,2-epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk-LEH and CH55-LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re-LEH) in terms of stereoselectivity. Tomsk-LEH and CH55-LEH, both from thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re-LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk-LEH and poly(ethylene glycol) for CH55-LEH. The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies. DatabaseThe atomic coordinates and structure factors of the crystal structures have been deposited in the Protein Data Bank with the codes 5AIF (Tomsk-LEH native structure), 5AIG (Tomsk-LEH valpromide complex), 5AIH (CH55-LEH native structure) and 5AII (CH55-LEH PEG complex). Nucleotide sequence data are available in the GenBank databases under the accession numbers KP765711 (Tomsk-LEH) and KP765710 (CH55-LEH).Abbreviations CH55-LEH, limonene-1,2-epoxide hydrolase from the metagenomic DNA from the Chinese sample; de, diastereomeric excess; ee, enantiomeric excess; EH, epoxide hydrolase; LEH, limonene-1,2-epoxide hydrolase; Mt-LEH, Mycobacterium tuberculosis limonene-1,2-epoxide hydrolase; PEG, poly(ethylene glycol); Re-LEH, Rhodococcus erythropolis limonene-1,2-epoxide hydrolase; Tomsk-LEH, limonene-1,2-epoxide hydrolase from the metagenomic DNA from the Tomsk sample.

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Section: Introductionmentioning

confidence: 99%

Discovery and characterization of thermophilic limonene‐1,2‐epoxide hydrolases from hot spring metagenomic libraries

et al. 2015

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“…Metagenomic or environmental DNA (eDNA), that is, the total microbial DNA of a microcosm, such as a small soil or groundwater sample, can serve as a source of novel EHs without the need to isolate and cultivate the microorganisms. PCR-based amplification of EH gene fragments in conjunction with genome-walking techniques [31] has been used to retrieve entire genes encoding α/β-hydrolase fold EHs directly from the metagenomic DNA [29,32]. Moreover, the activity Phylogenetic relationships among protein sequences encoding EHs with confirmed activities.…”

Section: Sources Of Ehsmentioning

confidence: 99%

“…(b) Determination of the regioselectivity coefficients α S and α R of the EH-catalyzed reaction using the wild-type and final evolved a Kau2 variant with racemic para-chlorostyrene oxide as the substrate. Plotting ee p against ee s · (1 − c) · c -1 enabled the determination of α R and subsequently α S as described in Kotik et al [32], using the intercept q and the slope m from the linear regression; for the calculations, nonabsolute values for ee p and ee s were used (see Moussou et al [58] reported using iterative saturation mutagenesis (Table 8.2; Figure 8.6). For example, higher levels of enantioconvergence toward racemic para-chlorostyrene oxide were achieved in only three productive mutagenesis rounds (Figure 8.6a), using a man ual two-step screen based on a colorimetric activity assay followed by a chiral GC test for ee p determination.…”

Section: Figure 85mentioning

confidence: 99%

“…These authors reported in the same article a sequential bienzymatic reaction using the wild-type EH from A. niger M200 and its evolved vari ant, resulting in the formation of the (R)-diol with an ee value of 88%. More recently, Kotik and coworkers reported that an EH (named Kau2), whose gene was iso lated from a biofilter-derived metagenome, exhibited an opposite regioselectivity for the two enantiomers of p-ClSO, which enabled them to obtain (R)-para-chlorophenyl-1,2-ethanediol in 84% ee at 100% conversion [32]. Later, an improvement in the enantio convergence of Kau2 was achieved using iterative saturation mutagenesis (Figure 8.6), leading to a higher enantiomeric excess of 94% for the diol product at complete con version of the racemic p-ClSO substrate [57].…”

Section: Chlorostyrene Oxidementioning

confidence: 99%

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Epoxide Hydrolases and their Application in Organic Synthesis

2016

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c h a p t e r 8 INTRODUCTIONOrganic chemists have become interested in enzymes as catalysts due to their high efficiencies and specificities. Moreover, recent progress in molecular biology and enzyme-related research areas enabled and simplified the production and purifica tion of recombinant enzymes in large quantities and their engineering toward tailormade biocatalysts using straightforward mutagenesis and screening techniques. This is also true for epoxide hydrolases (EHs), as evidenced by the many published research papers about the synthetic applications of naturally occurring or engi neered EHs. EHs catalyze the opening of oxirane rings, generating a vicinal diol as the final product ( Figure 8.1). From a synthetic chemistry point of view, the most valuable EHs are those with either (i) high enantioselectivities or (ii) a combination of low enantio preference with a high level of enantioconvergence. While the former generate enan tiopure epoxides with a maximum yield of 50% in a kinetic resolution process, the latter produce ideally an enantiopure diol product with a theoretical yield of 100%. Thus, EHs provide convenient access to enantiopure epoxides or diols from racemic epoxides. Furthermore, the enantiopure diols can then often be chemically trans formed back to the corresponding epoxides with no effect on the enantiomeric excess (ee). High values of enantioselectivity or enantioconvergence are a consequence of particular enzyme-substrate interactions, which can be modulated by specific reac tion conditions or through the exchange of specific amino acids of the biocatalyst. The final stereochemical outcome of an EH-catalyzed reaction depends solely on the regioselectivity coefficients, which determine the absolute configuration and the ee of the diol product when the reaction reaches 100% conversion. During the reaction, the oxirane ring of each enantiomer is often attacked at either carbon atom, resulting in a mixture of diol enantiomers (Figure 8.2). Unfortunately, several authors used the product-derived E-value, E P (calculated from c and ee P using Sih's equation; see Ref.[1]) for characterizing the stereochemistry of the diol formation of an EH-mediated hydrolysis reaction [2,3]. This is wrong and misleading for epoxide-opening reac tions since there are two possible positions of attack for each oxirane enantiomer. The ideal enantioconvergent EH leads to deracemization of a racemic mixture of an epox ide and exhibits reversed regioselectivity for either substrate enantiomer, that is,

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“…Enantiopure epoxides, a key block of bioactive compounds, are used to prepare the optically pure bioactive materials based on their high reactivity [1,2]. As one of several promising enantiopure epoxides, chiral epichlorohydrin has been widely used to prepare many chiral products, such as l-carnitine, pheromones, β-blockers, and ferroelectric liquid crystals [3,4].…”

Section: Introductionmentioning

confidence: 99%

Biosynthesis of (R)‐epichlorohydrin at high substrate concentration by kinetic resolution of racemic epichlorohydrin with a recombinant epoxide hydrolase

Jin

Liu

et al. 2013

Engineering in Life Sciences

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Research ArticleBiosynthesis of (R)-epichlorohydrin at high substrate concentration by kinetic resolution of racemic epichlorohydrin with a recombinant epoxide hydrolaseThe substrate concentration and yield were shown to be very low in the production of (R)-epichlorohydrin by hydrolysis of racemic epichlorohydrin using epoxide hydrolases in previous studies. In this work, we synthesized an epoxide hydrolase gene from Agrobacterium radiobacter and expressed it in Escherichia coli by the PCR assembly method. The recombinant A. radiobacter epoxide hydrolase (ArEH) was applied in the preparation of (R)-epichlorohydrin and, a yield of 42.7% with ≥99% enantiomeric excess (ee) from 25.6 mM racemic epichlorohydrin was obtained. However, the ee of (R)-epichlorohydrin was not able to reach 99% due to substrate and product inhibition when the substrate concentration was over 320 mM. Inhibition studies revealed that (S)-3-chloro-1,2-propanediol displayed noncompetitive inhibition in the conversion of (S)-epichlorohydrin but non-significant inhibition for (R)-epichlorohydrin. Moreover, ArEH was successfully applied in the preparation of (R)-epichlorohydrin at high substrate concentration by eliminating the substrate inhibition. The substrate concentration increased to 448 mM by intermittent feeding of the substrate and to 512 mM by using a two-phase reaction system, with a high yield (>27%) and ee (>98%) of (R)-epichlorohydrin. This is the first report of high-yield production of (R)-epichlorohydrin at high substrate concentration, laying the foundations for its application on the industrial scale.

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Access to enantiopure aromatic epoxides and diols using epoxide hydrolases derived from total biofilter DNA

Cited by 56 publications

References 45 publications

Discovery and characterization of thermophilic limonene‐1,2‐epoxide hydrolases from hot spring metagenomic libraries

Discovery and characterization of thermophilic limonene‐1,2‐epoxide hydrolases from hot spring metagenomic libraries

Epoxide Hydrolases and their Application in Organic Synthesis

Biosynthesis of (R)‐epichlorohydrin at high substrate concentration by kinetic resolution of racemic epichlorohydrin with a recombinant epoxide hydrolase

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