Sandbox Reserved 714
The Human soluble Epoxyde Hydroxylase (hsEH) is a homodimeric protein as well as a bifunctional enzyme, which is made of 555 aminoacids and can be found in the cytoplasm and in peroxisomes of humans. It belongs to the “Hydrolase” enzyme family. This enzyme has two functions: an epoxide hydrolase function on its C-term domain, which consists in the hydrolysis of epoxides into glycols, and a phosphatase function on its N-term domain, which consists in cleaving a phosphate group from a dihydroxy lipid phosphate.
Biological roleBiological role
This enzyme is expressed in mammalian liver, vascular endothelium, proximal tubule and some smooth muscles. By hydrolyzing epoxides and lipid phosphates, it has a role in detoxification and it prevents DNA from alkylation by those epoxides. At the physiological level, hsEH participates to the regulation of cardiovascular and renal physiology and to inflammatory biology as well[1]. More specifically, this enzyme is a regulator of mammalian blood pressure and could be a target for a treatment of hypertension[2]. Thanks to mutagenesis experiments[3], it has been demonstrated that the inhibition of sEH lowers blood pressure.
| General structure
The Human soluble Epoxide hydrolase is a protein of 555 residues. In vivo, it exists under the form of a homodimer, with a monomeric unit of 62,5 kDa. Each subunit has , linked by a proline-rich section. The secondary structure of this enzyme is made of beta-sheets (16 strands) and alpha-helices and a few 310-helices (34 helices), which form the two pockets of the active sites, in the C-term domain and the N-term domain, in which the substrates (lipid-phosphates and ions for the N-term domain, and epoxides for the C-term domain) can bind. The 3D dimensions of the enzyme are the following : a = 92.55 Å, b = 92.55 Å, c = 244.64 Å. The quaternary structure is called a domain-swapped dimer. The C-ter domain adopts an α/β-hydrolase fold, corresponding to its catalytic activity. Remarkably, the N-term domain has an α/β fold which is similar to HAD (Haloacid Dehalogenase) family. This N-term domain has no dehalogenase activity but a phosphatase one. The N-terminal domain has specific features that facilitate the binding of a lipid substrate. There are that ensure the proper positioning of the substrate. First, a hydrophobic cleft of about 25 Å long is situated near the N-term core so that one of the two ends of the aliphatic substrate is near the interface between the two domains N-term and C-term (proline-rich linker). Secondly, a hydrophobic tunnel (about 14 Å long) binds the aliphatic chain of the substrate and allows the second end to be in the active site. The active site is a negatively charged pocket of about 15 Å deep, and contains a Mg2+ cation necessary to the catalytic function. The aminoacids which are involved in the active site of the N-term domain are the Asp9, Asp11 and Asp185 basic aminoacids which can bind with a Mg2+ ion each, which is necessary to permit the substrate binding. These residues are characteristic of a significant part of phosphatases and phosphonatases. There is also a modified lysine, the Lys43, which has an acetyl group on its N6. The aminoacids which are involved in the active site of the C-term domain can be divided in two groups : the binding aminoacids and the catalytic aminoacids. The His239, Tyr241, Arg249 and Glu298 are involved in the binding of the substrate, the Hexaethylene Glycol (P6G). The Asp335, Asp496, Tyr466 and His524 are the catalytic aminoacids of the active site. Asp335 can lead nucleophilic additions, Tyr466 is a proton donor and His524 is a proton acceptor.
MechanismC-terminal domainThe C-terminal domain is called Cytosolic epoxide hydrolase 2: it catalyzes the trans-addition of water to epoxides in order to product glycols [4]. The corresponding reaction equation is the following: Epoxide + H2O ↔ Glycol The is made of five residues. The 3D structure of this active site is maintained by hydrogen bonds, including those created by D496. The two tyrosines (Y383 and Y466) assist the proper positioning of the substrate by polarizing it, thanks to their hydroxyl groups. D335 acts as a nucleophilic acid. Finally, H524 plays the role of a base in order to release the final product. The reaction proceeds in two steps, including the formation of a covalent intermediate. First, the substrate (epoxide) is accepted in the active site and its oxygen forms hydrogen bonds with Y383 and Y466. The oxygen of D335 attacks one of the two carbons included in the epoxide function. As a result, the oxygen of the subtrate takes the hydrogen of the hydroxyl function of Y466: the covalent intermediate is formed, and linked to D335. Then, the oxygen negatively charged belonging to the lateral chain of Y383 attacks the hydrogen of H524. After that, a water molecule enters the active site. The hydroxyl of D335 is therefore renewed, the diol product is created and released. The active site is available for a new catalytic cycle. N-terminal domainThe N-terminal domain is responsible of the Mg2+ dependant hydrolysis of dihydroxy lipid phosphates [5]. Indeed, the aliphatic substrate binds the protein on its hydrophobic tunnel, as it has been described previously. The specificity of this enzyme has been tested for several lipid molecules, and the best substrate found is the monophosphate of dihydroxy stearic acid (threo-9/10-phosphonoxy-hydroxy-octadecanoic acid) [6]. Indeed, the catalytic values for this substrate are Km = 21 +/- 0.3 μM, VMax = 338 +/- 12 nmol.min-1.mg-1, and kcat = 0.35 +/- 0.01 s-1. In the example of this substrate, the reaction follows this equation: 9/10-phosphonoxy-hydroxy-octadecanoate + H2O ↔ 9/10-dihydroxy-octadecanoate + phosphate Its contains several conserved aspartates in phosphatases and phosphonatases: D9, D11, D184 and D185. This enzymatic activity is Mg2+ dependant, because the structure of the active site is in its optimal conformation when the cation makes coordination interactions. When the catalytic activity of the N-term domain is available, Magnesium is octahedrally coordinated with the four aspartates, one water molecule and the phosphate belonging to the substrate. We can note that Mg2+ is not directly involved in the catalytic mechanism, and all its interactions with the active site remain during the hydrolysis. Its single role consists in maintaining the three-dimensional structure of the active site. First, the oxygen on the lateral chain of D9 attacks the phosphate. After the addition of a proton H+, the product with its two hydroxyl functions is released, while the phosphate is still linked to D9. Then, a waters molecule binds the phosphate, breaking its bond with the aspartate. Therefore the phosphate can be finally released and the active site can accept a lipid again and start a new catalytic cycle. InhibitorsAs many enzymes, the human soluble epoxide hydrolase can be inhibited by some inhibitors, causing a loss of activity for the enzyme. The sEH can be inhibited by some metal ions such as Zn2+, Cu2+, Hg2+ [4]. It is a noncompetitive inhibition: the metal ion binding to the enzyme reduces its activity without affecting directly the substrate binding, so that the Vmax decreases but the Km remains unchanged. It may be that those metal ions replace the Mg2+ in the N-term active site of the enzyme subunits, which can result in some conformation changes, but this is only an hypothesis. However, there are also some chemical inhibitors that inhibit the sEH. They are 1-3 disubstitued ureas, carbamates and amides, which are stable inhibitors for the sEH. Those chemical inhibitors are competitive inhibitors, which can bind in the active site of the enzyme in place of the right substrate, which graphically can be noticed by a Km increase, whereas the Vmax remains unchanged. In fact, according to crystal structures, urea can set up hydrogen bonds and salt bridges with some residues of the sEH active site, which ends up in an “enzyme-substrate”-like complex. Nevertheless, it is also important to notice that those inhibitor must have hydrophobic sidechains in order to fit in the hydrophobic zone of the catalytic site.
| ||||||||||
Additional 3D Structures of hsEHAdditional 3D Structures of hsEH
1vj5 - hsEH + N-cyclohexyl-N'-(4-iodophenyl)urea complex
1zd2,1zd3, 1zd4,1zd5 - hsEH + 4-(3-cyclohexyluriedo)-carboxylic acids
3ant - Hydrolase domain + synthetic inhibitor
3pdc - Hydrolase domain + benzoxazole inhibitor
External ressourcesExternal ressources
Protein Data Bank entry on 1S8O
Uniprot link on Bifunctional epoxyde hydrolase 2
Wikipedia page on Epoxyde hydrolase 2
ReferencesReferences
- ↑ Gomez GA, Morisseau C, Hammock BD, Christianson DW. Human soluble epoxide hydrolase: structural basis of inhibition by 4-(3-cyclohexylureido)-carboxylic acids. Protein Sci. 2006 Jan;15(1):58-64. Epub 2005 Dec 1. PMID:16322563 doi:10.1110/ps.051720206
- ↑ Sinal CJ, Miyata M, Tohkin M, Nagata K, Bend JR, Gonzalez FJ. Targeted disruption of soluble epoxide hydrolase reveals a role in blood pressure regulation. J Biol Chem. 2000 Dec 22;275(51):40504-10. PMID:11001943 doi:10.1074/jbc.M008106200
- ↑ Imig JD, Zhao X, Capdevila JH, Morisseau C, Hammock BD. Soluble epoxide hydrolase inhibition lowers arterial blood pressure in angiotensin II hypertension. Hypertension. 2002 Feb;39(2 Pt 2):690-4. PMID:11882632
- ↑ 4.0 4.1 Morisseau C, Hammock BD. Epoxide hydrolases: mechanisms, inhibitor designs, and biological roles. Annu Rev Pharmacol Toxicol. 2005;45:311-33. PMID:15822179 doi:10.1146/annurev.pharmtox.45.120403.095920
- ↑ Gomez GA, Morisseau C, Hammock BD, Christianson DW. Structure of human epoxide hydrolase reveals mechanistic inferences on bifunctional catalysis in epoxide and phosphate ester hydrolysis. Biochemistry. 2004 Apr 27;43(16):4716-23. PMID:15096040 doi:10.1021/bi036189j
- ↑ Newman JW, Morisseau C, Harris TR, Hammock BD. The soluble epoxide hydrolase encoded by EPXH2 is a bifunctional enzyme with novel lipid phosphate phosphatase activity. Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1558-63. Epub 2003 Feb 6. PMID:12574510 doi:10.1073/pnas.0437724100
Proteopedia Page Contributors and EditorsProteopedia Page Contributors and Editors
DUTREUX Fabien, BONHOURE Anna