Cytochrome P450/NADPH-dependent biosynthesis of 5,6- trans -epoxyeicosatrienoic acid from 5,6- trans -arachidonic acid

Human microsomal cytochrome P450 (CYP) 2E1 is widely known for its ability to oxidize >70 different, mostly compact, low molecular weight drugs and other xenobiotic compounds. In addition CYP2E1 oxidizes much larger C9 -C20 fatty acids that can serve as endogenous signaling molecules. Previously structures of CYP2E1 with small molecules revealed a small, compact CYP2E1 active site, which would be insufficient to accommodate medium and long chain fatty acids without conformational changes in the protein. In the current work we have determined how CYP2E1 can accommodate a series of fatty acid analogs by cocrystallizing CYP2E1 with -imidazolyl-octanoic fatty acid, -imidazolyl-decanoic fatty acid, and -imidazolyldodecanoic fatty acid. In each structure direct coordination of the imidazole nitrogen to the heme iron mimics the position required for native fatty acid substrates to yield the -1 hydroxylated metabolites that predominate experimentally. In each case rotation of a single Phe 298 side chain merges the active site with an adjacent void, significantly altering the active site size and topology to accommodate fatty acids. The binding of these fatty acid ligands is directly opposite the channel to the protein surface and the binding observed for fatty acids in the bacterial cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium. Instead of the BM3-like binding mode in the CYP2E1 channel, these structures reveal interactions between the fatty acid carboxylates and several residues in the F, G, and B helices at successive distances from the active site. Enzymes in the cytochrome P450 (CYP)2 superfamily are involved in the monooxygenation of a diverse repertoire of foreign and endogenous compounds. Of the 57 human enzymes in this superfamily, perhaps a third to a fourth are responsible for the metabolism of xenobiotics, including pharmaceuticals. However, a number of these enzymes also have putative roles in the metabolism of endogenous physiologically important compounds. Cytochrome P450 2E1 (CYP2E1) falls into this class.CYP2E1 is probably best known for its ability to oxidize xenobiotic compounds such as ethanol, the alcoholism treatment disulfiram (Antabuse), and the analgesic acetaminophen. In fact, the action of CYP2E1 on acetaminophen creates a metabolite responsible for hepatotoxicity and death in acetaminophen overdose cases. However, CYP2E1 is also known to oxidize endogenous fatty acids, including lipids associated with signaling mechanisms such as arachidonic acid (1) and epoxyeicosatrienoic acids (2). Specifically, biochemical investigations have revealed that CYP2E1 can bind a range of mid-length fatty acids and catalyze the (primarily -1) hydroxylation of saturated fatty acids and the epoxidation of the alkenes of unsaturated fatty acids. The metabolism of fatty acids by CYP may also be important in maintaining homeostasis of fatty acid levels. The role of CYP2E1 in the metabolism of these molecules may be related to its regulation by disease states such as obesity (3) and diabetes (3, 4) in rat mo...

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confidence: 99%

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confidence: 99%

Human Cytochrome P450 2E1 Structures with Fatty Acid Analogs Reveal a Previously Unobserved Binding Mode

Porubsky

Battaile

Scott

2010

“…These substrates include industrial solvents such as benzene, toluene, aniline, and halogenated solvents (18); alcohols such as ethanol (19), glycerol, phenol, and p-nitrophenol (20); and bicyclic heterocycles such as caffeine (21) and the muscle relaxant chlorzoxazone (22). However, CYP2E1 is also known to metabolize endogenous fatty acids, including lipids associated with signaling mechanisms such as arachidonic acid (23) and epoxyeicosatrienoic acids (24). The role of CYP2E1 in the metabolism of these signaling molecules may be related to its regulation by disease states such as obesity (25) and diabetes (26) in rat models.…”

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confidence: 99%

Structures of Human Cytochrome P-450 2E1

Porubsky¹,

Meneely²,

Scott

2008

195

102

Human microsomal cytochrome P-450 2E1 (CYP2E1) monooxygenates >70 low molecular weight xenobiotic compounds, as well as much larger endogenous fatty acid signaling molecules such as arachidonic acid. In the process, CYP2E1 can generate toxic or carcinogenic compounds, as occurs with acetaminophen overdose, nitrosamines in cigarette smoke, and reactive oxygen species from uncoupled catalysis. Thus, the diverse roles that CYP2E1 has in normal physiology, toxicity, and drug metabolism are related to its ability to metabolize diverse classes of ligands, but the structural basis for this was previously unknown. Structures of human CYP2E1 have been solved to 2.2 Å for an indazole complex and 2.6 Å for a 4-methylpyrazole complex. Both inhibitors bind to the heme iron and hydrogen bond to Thr 303 within the active site. Complementing its small molecular weight substrates, the hydrophobic CYP2E1 active site is the smallest yet observed for a human cytochrome P-450. The CYP2E1 active site also has two adjacent voids: one enclosed above the I helix and the other forming a channel to the protein surface. Minor repositioning of the Phe 478 aromatic ring that separates the active site and access channel would allow the carboxylate of fatty acid substrates to interact with conserved 216 QXXNN 220 residues in the access channel while positioning the hydrocarbon terminus in the active site, consistent with experimentally observed -1 hydroxylation of saturated fatty acids. Thus, these structures provide insights into the ability of CYP2E1 to effectively bind and metabolize both small molecule substrates and fatty acids.Cytochrome P-450 (P-450) 3 is a superfamily of enzymes involved in monooxygenation of both endogenous and exogenous substrates. A subset of these enzymes, including cytochrome P-450 2E1 (CYP2E1), are known for their role in the clearance of drugs and other xenobiotics by introducing or unmasking sites for subsequent conjugation and elimination from the body. From a biochemical standpoint these enzymes are fascinating for the diversity of substrates each xenobioticmetabolizing P-450 can metabolize, yet often with exquisite selectivity in the metabolites generated. Unfortunately, in the process some P-450-mediated metabolism can produce toxic or carcinogenic products. Among cytochrome P-450 enzymes, CYP2E1 is particularly notable for this ability and the resulting toxicity (1). This activity is most substantial in the liver because CYP2E1 comprises over 50% of the hepatic cytochrome P-450 mRNA (2) and 7% of the hepatic cytochrome P-450 protein (3). However, CYP2E1 is also expressed at lower levels in a variety of extrahepatic tissues (4), where it is thought to play a role in the metabolism of important endogenous molecules. CYP2E1 levels and the resulting toxicity varies markedly in response to alcohol consumption (5), diabetes (6), obesity (7), and fasting (8). Thus, the action of CYP2E1 can have a significant influence on human health and drug metabolism.CYP2E1 has been connected with liver toxicity through two ...

“…Because 13-H-14,15-EETA has a hydroxyl group separating the double bond and the epoxide group, it can be postulated that the hydroxyl group protects the epoxide from acid hydrolysis by a combination of steric shielding and inductive effects. Additionally, 5,6-trans-EET is more stable and resistant to hydrolysis than its cis-isomer (45). Thus, the trans-epoxide of 13-H-14,15-EETAs could also contribute to the acid stability.…”

Section: Discussionmentioning

confidence: 99%

Identification of 13-Hydroxy-14,15-epoxyeicosatrienoic Acid as an Acid-stable Endothelium-derived Hyperpolarizing Factor in Rabbit Arteries

Chawengsub

Gauthier

Nithipatikom

et al. 2009

Arachidonic acid (AA) is metabolized by endothelial 15-lipoxygenase (15-LO) to several vasodilatory eicosanoids such as 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) and its proposed unstable precursor 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). In the present study, the acid-stable 13-hydroxy-trans-14,15-epoxy-eicosatrienoic acid (13-H-14,15-EETA) was identified and its vascular activities characterized. Rabbit aorta, mesenteric arteries, and the combination of 15-LO and cytochrome P450 2J2 converted AA to two distinct HEETA metabolites. The HEETA metabolites were resistant to acidic hydrolysis but were hydrolyzed by recombinant sEH to a more polar metabolite identified by mass spectrometry as 13,14,15-THETA.