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

Abstract

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...

Nicotine and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Binding and Access Channel in Human Cytochrome P450 2A6 and 2A13 Enzymes

“…The reorientation of this single side chain nearly doubles the active site volume (598 Å 3 ) from that observed in previous structures containing a single ligand (ϳ300 Å 3 ). This type of Phe repositioning is very similar to two different structures of CYP2E1 where repositioning of either Phe-298 or Phe-478 on opposite sites of the active site can substantially increase the active site size to accommodate larger ligands (14,39). Simultaneous binding of more than one ligand has previously been observed for CYP3A4, CYP2C8, CYP2B4, and CYP21A2 (40 -43).…”

Human Liver Cytochrome P450 3A4 Ubiquitination

“…Furthermore, inspection of its crystal structure (33,94) also reveals that every one of its Lys residues ubiquitinated by these E2-E3 systems similarly resides within spatially associated and negatively charged Asp/Glu/ Ser(P)/Thr(P) surface clusters (30,76). It remains to be determined at present whether these are common structural features of the ubiquitination of not only all P450 proteins but also that of an expanding list of other canonical substrates of these particular E2-E3 ubiquitination complexes.…”

Human Cytochrome P450 1A1 Structure and Utility in Understanding Drug and Xenobiotic Metabolism

“…In addition, we compare and contrast the CYP1A1 active site residues and cavities with those of the other two members of the CYP1 family. Because other mammalian cytochrome P450 enzymes often have flexible active sites that depend on the ligand (32,33), we use docking to evaluate the practical question of whether this single structure of human CYP1A1 is sufficient to understand the binding of other ligands including substrates and inhibitors or whether further structures with different ligand scaffolds will be necessary to elucidate the full functional capacity of these enzymes. Finally, these docking outcomes were compared with docking of the same compounds to CYP1A2 to evaluate the implications of protein active site differences on ligand orientations and metabolism.…”