Gene_locus Report for: arath-At2g26740

Affinity chromatographic methods were developed for the one-step purification to homogeneity of recombinant soluble epoxide hydrolases (sEHs) from cress and potato. The enzymes are monomeric, with masses of 36 and 39 kDa and pI values of 4.5 and 5.0, respectively. In spite of a large difference in sequence, the two plant enzymes have properties of inhibition and substrate selectivity which differ only slightly from mammalian sEHs. Whereas mammalian sEHs are highly selective for trans- versus cis-substituted stilbene oxide and 1,3-diphenylpropene oxide (DPPO), plant sEHs exhibit far greater selectivity for trans- versus cis-stilbene oxide, but little to no selectivity for DPPO isomers. The isolation of a covalently linked plant sEH-substrate complex indicated that the plant and mammalian sEHs have a similar mechanism of action. We hypothesize an in vivo role for plant sEH in cutin biosynthesis, based on relatively high plant sEH activity on epoxystearate to form a cutin precursor, 9,10-dihydroxystearate. Plant sEHs display a high thermal stability relative to mammalian sEHs. This stability and their high enantioselectivity for a single substrate suggest that their potential as biocatalysts for the preparation of enantiopure epoxides should be evaluated.

Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130-140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.

A cDNA (1122 bp) was isolated from a cDNA library prepared from Arabidopsis thaliana L. that had been subjected to drought stress for 1 h. The sequencing of a genomic clone corresponding to the cDNA and S1 mapping analysis revealed that the cDNA lacked the first 6 bp from its translational start (ATG). The resulting open reading frame encodes a polypeptide of 321 amino acids, and the calculated molecular weight of this polypeptide is 36,423 Da. The deduced amino acid sequence shows a high degree of similarity to C terminal halves of those of soluble epoxide hydrolases (sEHs) of human, mouse and rat, 35.5%, 34.1% and 33.1%, respectively. The cDNA was expressed in Escherichia coli cells, and the expressed protein migrates at 40 kDa when analyzed by SDS-PAGE. The recombinant protein at 40 kDa is much smaller than the mammalian sEH (58 kDa) but has characteristics of activity and inhibition similar to the mammalian sEHs when assayed with the substrate trans-stilbene oxide and the inhibitors 4-fluorochalcone oxide (4FCO), (2R,3R)-3-(4-nitrophenyl) glycidol (RRNPG), and (2S,3S)-3-(4-nitrophenyl)glycidol (SSNPG), which indicates that the cDNA did encode a soluble epoxide hydrolase of A. thaliana (AtsEH). Drought stress, but not heat or cold stress, slightly increased the accumulation of the mRNAs for AtsEH. The level of AtsEH transcripts increased strongly after treatment with a plant hormone, auxin (2,4-dichlorophenoxyacetic acid, 2,4-D; naphthalene-acetic acid, NAA; and indole-3-acetic acid, IAA) in young, pre-bolting plants. Treatment with cytokinin (6-benzylaminopurine, BA), abscisic acid (ABA) or gibberellin (GA3) had no detectable effect on AtsEH transcript levels. The transcripts for AtsEH gene were detected in the aerial vegetative organs of bolting plants (i.e. stems and leaves), but not in roots, flowers and seeds. The possible function of AtsEH is discussed. A similar sEH cDNA has recently been characterized in potato (Stapleton et al., 1994).

Epoxide hydrolases (EHs) are present in all living organisms. They have been extensively characterized in mammals; however, their biological functions in plants have not been demonstrated. Based on in silico analysis, we identified AtEH1 (At3g05600), a putative Arabidopsis thaliana epoxide hydrolase possibly involved in cutin monomer synthesis. We expressed AtEH1 in yeast and studied its localization in vivo. We also analyzed the composition of cutin from A. thaliana lines in which this gene was knocked out. Incubation of recombinant AtEH1 with epoxy fatty acids confirmed its capacity to hydrolyze epoxides of C18 fatty acids into vicinal diols. Transfection of Nicotiana benthamiana leaves with constructs expressing AtEH1 fused to enhanced green fluorescent protein (EGFP) indicated that AtEH1 is localized in the cytosol. Analysis of cutin monomers in loss-of-function Ateh1-1 and Ateh1-2 mutants showed an accumulation of 18-hydroxy-9,10-epoxyoctadecenoic acid and a concomitant decrease in corresponding vicinal diols in leaf and seed cutin. Compared with wild-type seeds, Ateh1 seeds showed delayed germination under osmotic stress conditions and increased seed coat permeability to tetrazolium red. This work reports a physiological role for a plant EH and identifies AtEH1 as a new member of the complex machinery involved in cutin synthesis.

Epoxide hydrolases are enzymes involved in metabolism and defense of plants. Genome scanning suggested the presence of several genes encoding epoxide hydrolase in Arabidopsis thaliana. To assure that the predicted genes are functional and the translated products have epoxide hydrolase activity analysis at the protein level is needed. We have started to clone the cDNAs and overexpress them for catalytic and physico-chemical analysis. We here report that Pichia pastoris serves as an efficient system for overexpression of soluble epoxide hydrolase 1 (AtsEH1) from A. thaliana. A tag containing six histidine residues was added to the N-terminus to enable efficient one-step purification on nickel-agarose. The enzyme was expressed at levels >18 mg.L(-1) of culture and a French Press was found to be effective to achieve cell lysis. The recombinant enzyme had a molecular mass of 37 or 38 kDa based on SDS-PAGE or MALDI-TOF analysis, respectively. The enzyme was highly active towards the substrate trans-stilbene oxide (TSO) and had a pH optimum at 7 and a temperature optimum at 54 degrees C. Using TSO as substrate the K(m) and V(max) values were determined to 5 micro M and 2 micromol min(-1) mg protein(-1), respectively. The activity was 50-fold lower towards cis-stilbene oxide. The stability over time was tested from 20 to 54 degrees C and the enzyme lost activity at varying degrees at the temperatures tested but was stable for several months at 4 degrees C.

Affinity chromatographic methods were developed for the one-step purification to homogeneity of recombinant soluble epoxide hydrolases (sEHs) from cress and potato. The enzymes are monomeric, with masses of 36 and 39 kDa and pI values of 4.5 and 5.0, respectively. In spite of a large difference in sequence, the two plant enzymes have properties of inhibition and substrate selectivity which differ only slightly from mammalian sEHs. Whereas mammalian sEHs are highly selective for trans- versus cis-substituted stilbene oxide and 1,3-diphenylpropene oxide (DPPO), plant sEHs exhibit far greater selectivity for trans- versus cis-stilbene oxide, but little to no selectivity for DPPO isomers. The isolation of a covalently linked plant sEH-substrate complex indicated that the plant and mammalian sEHs have a similar mechanism of action. We hypothesize an in vivo role for plant sEH in cutin biosynthesis, based on relatively high plant sEH activity on epoxystearate to form a cutin precursor, 9,10-dihydroxystearate. Plant sEHs display a high thermal stability relative to mammalian sEHs. This stability and their high enantioselectivity for a single substrate suggest that their potential as biocatalysts for the preparation of enantiopure epoxides should be evaluated.

Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130-140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.

A cDNA (1122 bp) was isolated from a cDNA library prepared from Arabidopsis thaliana L. that had been subjected to drought stress for 1 h. The sequencing of a genomic clone corresponding to the cDNA and S1 mapping analysis revealed that the cDNA lacked the first 6 bp from its translational start (ATG). The resulting open reading frame encodes a polypeptide of 321 amino acids, and the calculated molecular weight of this polypeptide is 36,423 Da. The deduced amino acid sequence shows a high degree of similarity to C terminal halves of those of soluble epoxide hydrolases (sEHs) of human, mouse and rat, 35.5%, 34.1% and 33.1%, respectively. The cDNA was expressed in Escherichia coli cells, and the expressed protein migrates at 40 kDa when analyzed by SDS-PAGE. The recombinant protein at 40 kDa is much smaller than the mammalian sEH (58 kDa) but has characteristics of activity and inhibition similar to the mammalian sEHs when assayed with the substrate trans-stilbene oxide and the inhibitors 4-fluorochalcone oxide (4FCO), (2R,3R)-3-(4-nitrophenyl) glycidol (RRNPG), and (2S,3S)-3-(4-nitrophenyl)glycidol (SSNPG), which indicates that the cDNA did encode a soluble epoxide hydrolase of A. thaliana (AtsEH). Drought stress, but not heat or cold stress, slightly increased the accumulation of the mRNAs for AtsEH. The level of AtsEH transcripts increased strongly after treatment with a plant hormone, auxin (2,4-dichlorophenoxyacetic acid, 2,4-D; naphthalene-acetic acid, NAA; and indole-3-acetic acid, IAA) in young, pre-bolting plants. Treatment with cytokinin (6-benzylaminopurine, BA), abscisic acid (ABA) or gibberellin (GA3) had no detectable effect on AtsEH transcript levels. The transcripts for AtsEH gene were detected in the aerial vegetative organs of bolting plants (i.e. stems and leaves), but not in roots, flowers and seeds. The possible function of AtsEH is discussed. A similar sEH cDNA has recently been characterized in potato (Stapleton et al., 1994).