The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein

Friedberg, Thomas; Löllmann, Bettina; Becker, Roger; Holler, Romy; Oesch, Franz

doi:10.1042/bj3030967

Cited by 39 publications

(24 citation statements)

References 34 publications

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“…Its members are, on average, 100 (bacterial), 120 (fungal), and 150 (higher eukaryotes) amino acids longer than most other epoxide hydrolases (Table 2), which is mainly due to a large N-terminal extension. The mammalian and insect epoxide hydrolases of this group are membrane bound by an N-terminal membrane anchor (20). The fungal and bacterial epoxide hydrolases of this group do not contain this membrane anchor and are therefore probably soluble.…”

Section: Resultsmentioning

confidence: 99%

Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis

Loo

Kingma

Arand

et al. 2006

Appl Environ Microbiol

109

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Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative ␣/␤-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts.Enantiopure epoxides and vicinal diols are valuable intermediates in the synthesis of a number of pharmaceutical compounds. Epoxide hydrolases (EC 3.3.2.3) catalyze the conversion of epoxides to the corresponding diols. If they are enantioselective, they can be used to produce enantiopure epoxides by means of kinetic resolution (5). In the past, when only epoxide hydrolases from mammalian sources were known (12), the use of epoxide hydrolases in biocatalysis was hampered by their poor availability and insufficient catalytic performance, such as a low turnover rate or poor enantioselectivity. The potential for biocatalytic application of epoxide hydrolases was significantly increased with the discovery of microbial epoxide hydrolases (41), which are easier to produce in large quantities. The cloning and overexpression of several enantioselective epoxide hydrolases, e.g., from Agrobacterium radiobacter (35), Aspergillus niger (3), and potato plants (40), not only facilitated large-scale production of these enzymes but also made it possible to improve their biocatalytic properties by site-directed or random mutagenesis (34,36,43).Since many microbial genome sequences are available in the public domain, it is useful to screen these databases for genes that might encode new enzymes with interesting properties.Novel epoxide hydrolases can be identified by performing a BLAST search of the genomic databases, using amino acid sequences of known epoxide hyd...

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Section: Resultsmentioning

confidence: 99%

Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis

Loo

Kingma

Arand

et al. 2006

Appl Environ Microbiol

109

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“…In contrast, release of VKOR from the ER membrane required detergent (squares). The detergent concentrations used were also shown to release the two ER integral membrane proteins mGST and mEH, which are both anchored to the membrane by one transmembrane domain (28,29). This shows that some of the VKOR protein components are firmly attached to the ER membrane.…”

Section: Association Of Cytosolic Gsts and Vkor With The Er Membrane-mentioning

confidence: 91%

Assembly of the Warfarin-sensitive Vitamin K 2,3-Epoxide Reductase Enzyme Complex in the Endoplasmic Reticulum Membrane

Cain

Hutson

Wallin

1997

Journal of Biological Chemistry

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␥-Carboxylation of vitamin K-dependent proteins requires a functional vitamin K cycle to produce the active vitamin K cofactor for the ␥-carboxylase which posttranslationally modifies precursors of these proteins to contain ␥-carboxyglutamic acid residues. The warfarinsensitive enzyme vitamin K epoxide reductase (VKOR) of the cycle reduces vitamin K 2,3-epoxide to the active vitamin K hydroquinone cofactor. Because of the importance of warfarin as an anticoagulant in prophylactic medicine and as a poison in rodent pest control, numerous attempts have been made to understand the molecular mechanism underlying warfarin-sensitive vitamin K 2,3-epoxide reduction. In search for protein components that could be involved in this reaction we designed an in vitro ␥-carboxylation test system where the warfarin-sensitive VKOR produces the cofactor for the ␥-carboxylase. Dissection of this system by chromatographic techniques has identified a member(s) of the glutathione S-transferase gene family as one component of the VKOR enzyme complex in the endoplasmic reticulum membrane. The affinity-purified glutathione Stransferase(s) was sensitive to warfarin but lost its warfarin sensitivity and glutathione S-transferase activity upon association with lipids in the presence of Mn 2؉ or Ca 2؉. In the ␥-carboxylation test system, loss of warfarin-sensitive glutathione S-transferase activity coincided with formation of the VKOR enzyme complex. It is proposed that formation of VKOR in the endoplasmic reticulum membrane resembles formation of the lipoxygenase enzyme complex where the glutathione S-transferase-related FLAP protein binds cytosolic lipoxygenase to form a membrane enzyme complex.

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“…Hydropathy analysis has suggested the presence of 4 (11) or 6 (31) domains that integrate into the membrane. Other studies, however, have suggested the existence of only a single transmembrane domain comprising amino acids 4 -20 (32,33). The cDNA of mEHt was transfected into COS-7 cells, and analysis of total protein indicated the synthesis of a protein at approximately the same level of expression as the other derivatives in this study, with the predicted decrease in molecular weight (Fig.…”

Section: Fig 4 Expression Of Meh and Meh Derivatives In Cos-7 Cellsmentioning

confidence: 97%

“…Hydropathy analysis indicates the presence of a hydrophobic N-terminal region as well as several additional domains that could integrate into the membrane (11,31). Studies using truncated mEH suggested that mEH has only a single transmembrane domain, based on the results of alkaline extraction of membranes containing mEH or the truncated derivative (32). The expression of this truncated derivative, however, was only 5% of the level observed for mEH in BHK cells, and the authors failed to point out that approximately 40% of the truncated mEH appeared to be resistant to this extraction procedure, thereby leaving the conclusion of this study open to question.…”

Section: Discussionmentioning

confidence: 99%

Membrane Topology and Cell Surface Targeting of Microsomal Epoxide Hydrolase

Zhu¹,

Dippe²,

Xing³

et al. 1999

Journal of Biological Chemistry

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Microsomal epoxide hydrolase (mEH) is a bifunctional membrane protein that plays a central role in the metabolism of xenobiotics and in the hepatocyte uptake of bile acids. Numerous studies have established that this protein is expressed both in the endoplasmic reticulum and at the sinusoidal plasma membrane. Preliminary evidence has suggested that mEH is expressed in the endoplasmic reticulum (ER) membrane with two distinct topological orientations. To further characterize the membrane topology and targeting of this protein, an N-glycosylation site was engineered into mEH to serve as a topological probe for the elucidation of the cellular location of mEH domains. The cDNAs for mEH and this mEH derivative (mEHg) were then expressed in vitro and in COS-7 cells. Analysis of total expressed protein in these systems indicated that mEHg was largely unglycosylated, suggesting that expression in the ER was primarily of a type I orientation (Ccyt/Nexo). However, analysis, by biotin/avidin labeling procedures, of mEHg expressed at the surface of transfected COS-7 cells, showed it to be fully glycosylated, indicating that the topological form targeted to this site originally had a type II orientation (Cexo/Ncyt) in the ER. The surface expression of mEH was also confirmed by confocal fluorescence scanning microscopy. The sensitivity of mEH topology to the charge at the N-terminal domain was demonstrated by altering the net charge over a range of 0 to ؉3. The introduction of one positive charge led to a significant inversion in mEH topology based on glycosylation site analysis. A truncated form of mEH lacking the N-terminal hydrophobic transmembrane domain was also detected on the extracellular surface of transfected COS-7 cells, demonstrating the existence of at least one additional transmembrane segment. These results suggest that mEH may be integrated into the membrane with multiple transmembrane domains and is inserted into the ER membrane with two topological orientations, one of which is targeted to the plasma membrane where it mediates bile acid transport.Hepatic microsomal epoxide hydrolase (mEH) 1 (EC 3.3.2.3) is expressed in the endoplasmic reticulum (ER) membrane, where it is involved in the metabolism of many xenobiotics such as polycyclic aromatic hydrocarbon carcinogens (1), in concert with other proteins such as members of the cytochrome P450 enzyme superfamily (2, 3), dihydrodiol dehydrogenase (4), and glutathione S-transferase (5). Numerous studies (6 -12) have established that this protein is also expressed at the plasma membrane where it is able to mediate the sodium-dependent uptake of bile acids. The bile acids play an important role in many physiological processes such as (a) digestion; (b) the formation of bile, which functions as an excretory vehicle for numerous compounds such as cholesterol and metabolites of drugs and carcinogens; (c) the regulation of cholesterol metabolism; and (d) the modulation of hepatocyte signaling pathways. The surface location of mEH was initially suggested by enzyme m...

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The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein

Cited by 39 publications

References 34 publications

Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis

Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis

Assembly of the Warfarin-sensitive Vitamin K 2,3-Epoxide Reductase Enzyme Complex in the Endoplasmic Reticulum Membrane

Membrane Topology and Cell Surface Targeting of Microsomal Epoxide Hydrolase

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