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Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids.

ABSTRACT: Human cytochrome P450 (P450) 2W1 is still considered an "orphan" because its physiological function is not characterized. To identify its substrate specificity, the purified recombinant enzyme was incubated with colorectal cancer extracts for untargeted substrate searches using an LC/MS-based metabolomic and isotopic labeling approach. In addition to previously reported fatty acids, oleyl (18:1) lysophosphatidylcholine (LPC, lysolecithin) was identified as a substrate for P450 2W1. Other human P450 enzymes tested showed little activity with 18:1 LPC. In addition to the LPCs, P450 2W1 acted on a series of other lysophospholipids, including lysophosphatidylinositol, lysophosphatidylserine, lysophosphatidylglycerol, lysophosphatidylethanolamine, and lysophosphatidic acid but not diacylphospholipids. P450 2W1 utilized sn-1 18:1 LPC as a substrate much more efficiently than the sn-2 isomer; we conclude that the sn-1 isomers of lysophospholipids are preferred substrates. Chiral analysis was performed on the 18:1 epoxidation products and showed enantio-selectivity for formation of (9R,10S) over (9S,10R). [corrected]. The kinetics and position specificities of P450 2W1-catalyzed oxygenation of lysophospholipids (16:0 LPC and 18:1 LPC) and fatty acids (C16:0 and C18:1) were also determined. Epoxidation and hydroxylation of 18:1 LPC are considerably more efficient than for the C18:1 free fatty acid.

SUBMITTER: Xiao Y

PROVIDER: S-EPMC3540858 | biostudies-other | 2012 Aug

REPOSITORIES: biostudies-other

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Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids.

Xiao Yi Y Guengerich F Peter FP

Journal of lipid research 20120516 8

Human cytochrome P450 (P450) 2W1 is still considered an "orphan" because its physiological function is not characterized. To identify its substrate specificity, the purified recombinant enzyme was incubated with colorectal cancer extracts for untargeted substrate searches using an LC/MS-based metabolomic and isotopic labeling approach. In addition to previously reported fatty acids, oleyl (18:1) lysophosphatidylcholine (LPC, lysolecithin) was identified as a substrate for P450 2W1. Other human P ...[more]

PMID: 22591743

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Project description:BackgroundCytochrome P450s (CYPs) are important heme-containing proteins, well known for their monooxygenase reaction. The human cytochrome P450 4X1 (CYP4X1) is categorized as "orphan" CYP because of its unknown function. In recent studies it is found that this enzyme is expressed in neurovascular functions of the brain. Also, various studies have found the expression and activity of orphan human cytochrome P450 4X1 in cancer. It is found to be a potential drug target for cancer therapy. However, three-dimensional structure, the active site topology and substrate specificity of CYP4X1 remain unclear.MethodsIn the present study, the three-dimensional structure of orphan human cytochrome P450 4X1 was generated by homology modeling using Modeller 9v8. The generated structure was accessed for geometrical errors and energy stability using PROCHECK, VERFIY 3D and PROSA. A molecular docking analysis was carried out against substrates arachidonic acid and anandamide and the docked substrates were predicted for drug-likeness, ADME-Tox parameters and biological spectrum activity.ResultsThe three-dimensional model of orphan human cytochrome P450 4X1 was generated and assessed with various structural validation programmes. Docking of orphan human cytochrome P450 4X1 with arachidonic acid revealed that TYR 112, ALA 126, ILE 222, ILE 223, THR 312, LEU 315, ALA 316, ASP 319, THR 320, PHE 491 and ILE 492 residues were actively participating in the interaction, while docking of CYP4X1 with anandamide showed that TYR 112, GLN 114, PRO 118, ALA 126, ILE 222, ILE 223, SER 251, LEU 315, ALA 316 and PHE 491 key residues were involved in strong interaction.ConclusionFrom this study, several key residues were identified to be responsible for the binding of arachidonic acid and anandamide with orphan human cytochrome P450 4X1. Both substrates obeyed Lipinski rule of five in drug-likeness test and biological spectrum prediction showed anticarcinogenic activity. Compared to anandamide, arachidonic acid showed strong interaction with cytochrome P450 4X1 and also less health effect in certain human system in ADME-Tox prediction. These findings provide useful information on the biological role and structure-based drug design of orphan human cytochrome P450 4X1.

Project description:Selective oxidation of C-H bonds in alkylphenols holds great significance for not only structural derivatization in pharma- and biomanufacturing but also biological degradation of these toxic chemicals in environmental protection. A unique chemomimetic biocatalytic system using enzymes from a p-cresol biodegradation pathway has recently been developed. As the central biocatalyst, the cytochrome P450 monooxygenase CreJ oxidizes diverse p- and m-alkylphenol phosphates with perfect stereoselectivity at different efficiencies. However, the mechanism of regio- and stereoselectivity of this chemomimetic biocatalytic system remained unclear. Here, using p- and m-ethylphenol substrates, we elucidate the CreJ-catalyzed key steps for selective oxidations. The crystal structure of CreJ in complex with m-ethylphenol phosphate was solved and compared with its complex structure with p-ethylphenol phosphate isomer. The results indicate that the conformational changes of substrate-binding residues are slight, while the substrate promiscuity is achieved mainly by the available space in the catalytic cavity. Moreover, the catalytic preferences of regio- and stereoselective hydroxylation for the two ethylphenol substrates is explored by molecular dynamics simulations. The ethyl groups in the complexes display different flexibilities, and the distances of the active oxygen to H pro-S and H pro-R of methylene agree with the experimental stereoselectivity. The regioselectivity can be explained by the distances and bond dissociation energy. These results provide not only the mechanistic insights into CreJ regio- and stereoselectivity but also the structural basis for further P450 enzyme design and engineering.IMPORTANCE The key cytochrome P450 monooxygenase CreJ showed excellent regio- and stereoselectivity in the oxidation of various alkylphenol substrates. C-H bond functionalization of these toxic alkylphenols holds great significance for both biological degradation of these environmental chemicals and production of value-added structural derivatives in pharmaceutical and biochemical industries. Our results, combined with in vitro enzymatic assays, crystal structure determination of enzyme-substrate complex, and molecular dynamics simulations, provide not only significant mechanism elucidation of the regio- and stereoselective catalyzation mediated by CreJ but also the promising directions for future engineering efforts of this enzyme toward more useful products. It also has great extendable potential to couple this multifunctional P450 enzyme with other biocatalysts (e.g., hydroxyl-based glycosylase) to access more alkylphenol-derived high-value chemicals through environment-friendly biocatalysis and biotransformation.

Dataset Information

Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids.

Publications

Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids.

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