Microbial Epoxide Hydrolases.

Faber, Kurt; Mischitz, Martin; Kroutil, Wolfgang; Roos, Eric C.; Broxterman, Quirinus B.; Tweel, W.J.J. van den; Kamphuis, J.

doi:10.3891/acta.chem.scand.50-0249

Cited by 91 publications

(28 citation statements)

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“…The second identified protein of this group, the epoxide hydrolase, has been described as a protein that is essential for utilization of alkenes as carbon sources in prokaryotes (14). Alkenes are common compounds that exist in a variety of chemical forms and may be found in the root exudates of plants (35).…”

Section: Resultsmentioning

confidence: 99%

Identification of Streptomyces coelicolor Proteins That Are Differentially Expressed in the Presence of Plant Material

Langlois

Bourassa

Poirier

et al. 2003

Appl Environ Microbiol

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Streptomyces coelicolor and Lemna minor were used as a model to study the modulation of bacterial gene expression during plant-streptomycete interactions. S. coelicolor was grown in minimal medium with and without L. minor fronds. Bacterial proteomes were analyzed by two-dimensional gel electrophoresis, and a comparison of the two culture conditions resulted in identification of 31 proteins that were induced or repressed by the presence of plant material. One-half of these proteins were identified by peptide mass fingerprinting by using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The induced proteins were involved in energetic metabolism (glycolysis, pentose phosphate pathway, oxidative phosphorylation), protein synthesis, degradation of amino acids, alkenes, or cellulose, tellurite resistance, and growth under general physiological or oxidative stress conditions. The repressed proteins were proteins synthesized under starvation stress conditions. These results suggest that root exudates provide additional carbon sources to the bacteria and that physiological adaptations are required for efficient bacterial growth in the presence of plants.Plant rhizospheres harbor diverse communities of microorganisms. It is generally assumed that rhizosphere microbes use compounds released by the plant roots as their major nutrient sources. This ability is the nutritional basis of rhizosphere competence (32). Streptomycetes are gram-positive bacteria that are frequently isolated from terrestrial plant rhizospheres (3, 15), but Wohl and McArthur (49) also showed that streptomycetes are associated with freshwater plants. Until now, few research efforts have been dedicated to the study of plantstreptomycete interactions, and most previous efforts have involved the common scab-inducing streptomycetes and their host plants (29). As several Streptomyces species have been shown to be effective biocontrol agents for plant diseases (10), studies on the interactions between saprophytic streptomycetes and plants need to be documented more.We propose Streptomyces coelicolor and Lemna minor as an experimental model to study the modulation of bacterial gene expression during the interaction between a saprophytic streptomycete and a plant. Recent research in our laboratory has shown that L. minor, a small aquatic plant extensively used in bioremediation (38) and environmental studies (30), is colonized by various Streptomyces species (unpublished data). On the other hand, S. coelicolor is a common inhabitant of plant rhizospheres (11) that can be readily cocultivated in the presence of L. minor under sterile conditions. The fact that the S. coelicolor chromosome (6) has been fully sequenced is an important asset in proteomic studies.Proteomics has become an integral part of gene expression analysis. The functional complement of genetic information can be analyzed by a combination of two-dimensional gel electrophoresis for the separation of complex mixtures of proteins and mass spectrometry for identification...

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Section: Resultsmentioning

confidence: 99%

Identification of Streptomyces coelicolor Proteins That Are Differentially Expressed in the Presence of Plant Material

Langlois

Bourassa

Poirier

et al. 2003

Appl Environ Microbiol

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“…Although mammalian EHs have been extensively studied for detoxification, interest in microbial EHs has arisen primarily because of the potential of the enzymes as enantioselective biocatalysts (1,7,8). It is also of interest to investigate the detoxification role of microbial EHs and genetically adapt this universally successful detoxification strategy to the process of aerobic, cometabolic biodegradation of chlorinated ethenes in which toxic epoxides form as the primary intermediates (6).…”

Section: (6-fold)mentioning

confidence: 99%

Active Site Engineering of the Epoxide Hydrolase from Agrobacterium radiobacter AD1 to Enhance Aerobic Mineralization of cis-1,2-Dichloroethylene in Cells Expressing an Evolved Toluene ortho-Monooxygenase

Rui

Chen

et al. 2004

Journal of Biological Chemistry

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Chlorinated ethenes are the most prevalent groundwater pollutants, and the toxic epoxides generated during their aerobic biodegradation limit the extent of transformation. Hydrolysis of the toxic epoxide by epoxide hydrolases represents the major biological detoxification strategy; however, chlorinated epoxyethanes are not accepted by known bacterial epoxide hydrolases. Here, the epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA), which enables growth on epichlorohydrin, was tuned to accept cis-1,2-dichloroepoxyethane as a substrate by accumulating beneficial mutations from three rounds of saturation mutagenesis at three selected active site residues, Phe-108, Ile-219, and Cys-248 (no beneficial mutations were found at position Ile-111). The EchA F108L/I219L/C248I variant coexpressed with a DNA-shuffled toluene ortho-monooxygenase, which initiates attack on the chlorinated ethene, enhanced the degradation of cis-dichloroethylene (cis-DCE) an infinite extent compared with wildtype EchA at low concentrations (6.8 M) and up to 10-fold at high concentrations (540 M). EchA variants with single mutations (F108L, I219F, or C248I) enhanced cis-DCE mineralization 2.5-fold (540 M), and EchA variants with double mutations, I219L/C248I and F108L/C248I, increased cis-DCE mineralization 4-and 7-fold, respectively (540 M). For complete degradation of cis-DCE to chloride ions, the apparent V max /K m for the recombinant Escherichia coli strain expressing the EchA F108L/ I219L/C248I variant was increased over 5-fold as a result of the evolution of EchA. The EchA F108L/I219L/C248I variant also had enhanced activity for 1,2-epoxyhexane (2-fold) and the natural substrate epichlorohydrin (6-fold).

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“…Epoxide hydrolases from microbial sources have been increasingly recognized as highly versatile biocatalysts for the preparation of enantiopure epoxides and vicinal diols (6,33,37). Despite the excellent enantioselectivity found with certain substrates, the enantioselectivity of microbial epoxide hydrolases is in general often low (6).…”

Section: Discussionmentioning

confidence: 99%

“…Whereas mammalian EHs have been extensively studied for their important role in the detoxification of epoxides derived from xenobiotics (1), their use as biocatalysts on a preparative scale has been impeded by the lack of availability of the enzymes (6). Recently, with the identification of more EHs from microbial sources which have excellent enantioselectivity (2), and given that they may be produced on an almost unlimited scale (6), interest has increased in using microbial EHs for preparing epoxides or vicinal diols at high enantiomeric purity (6,33,37).…”

mentioning

confidence: 99%

Protein Engineering of Epoxide Hydrolase from Agrobacterium radiobacter AD1 for Enhanced Activity and Enantioselective Production of ( R )-1-Phenylethane-1,2-Diol

Rui

Chen

et al. 2005

Appl Environ Microbiol

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DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of Agrobacterium radiobacter AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (R)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (R)-1-phenylethane-1,2-diol (1.96 ؎ 0.09 versus 1.04 ؎ 0.07 mol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (R)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (R) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold-and 2-fold-increased activity on 5 mM epoxypropane (24 ؎ 2 versus 2.4 ؎ 0.3 mol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 ؎ 0.5 versus 2.6 ؎ 0.0 mol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 ؎ 0.3 versus 1.8 ؎ 0.2 mol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 ؎ 0.8 versus 1.8 ؎ 0.2 mol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.

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Microbial Epoxide Hydrolases.

Cited by 91 publications

References 0 publications

Identification of Streptomyces coelicolor Proteins That Are Differentially Expressed in the Presence of Plant Material

Identification of Streptomyces coelicolor Proteins That Are Differentially Expressed in the Presence of Plant Material

Active Site Engineering of the Epoxide Hydrolase from Agrobacterium radiobacter AD1 to Enhance Aerobic Mineralization of cis-1,2-Dichloroethylene in Cells Expressing an Evolved Toluene ortho-Monooxygenase

Protein Engineering of Epoxide Hydrolase from Agrobacterium radiobacter AD1 for Enhanced Activity and Enantioselective Production of ( R )-1-Phenylethane-1,2-Diol

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