title: Elucidation of functions of human cytochrome P450 enzymes: identification of endogenous substrates in tissue extracts using metabolomic and isotopic labeling approaches.
abstract: One of the central problems in biochemistry in the postgenomic era is the elucidation of functions of proteins, including "orphan" human cytochromes P450 (P450s), when the substrates are unknown. A general strategy for identification of endogenous substrates of P450s in tissue extracts using metabolomic and isotopic labeling approaches is described, involving four main steps: (1) In vitro incubation of a P450 enzyme system with cofactor and tissue extract is done under a mixture of (18)O(2)/(16)O(2) (1:1). (2) Liquid chromatography/mass spectrometry (LC/MS) assay of an organic extract of the reaction mixture is performed. (3) The isotopic labeling products appearing as M/M + 2 doublets can be directly identified using the program DoGEX (Sanchez-Ponce, R. and Guengerich, F. P. Anal. Chem. 2007, 79, 3355-3362). (4) Characterization of potential candidates is done. Validation of the strategy was established using human P450 7A1 as an initial model to identify its known product, 7alpha-hydroxycholesterol, in liver extracts. The strategy was then applied to human P450s 1A2, 2C8, and 2C9 in untargeted substrate searches with human liver extracts. A total of seven fatty acids were identified and verified as substrates of these three hepatic P450s. The products were subsequently characterized as hydroxylation and epoxidation derivatives of fatty acids, using gas chromatography/mass spectrometry (GC/MS) analysis. Finally, kinetic studies were performed to confirm that the fatty acids are oxidized by P450s 1A2, 2C8, and 2C9. Thus, this strategy has been demonstrated to be useful in identifying reactions in tissue extracts with orphan human P450s.
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 2200 Pierce Avenue, Nashville, Tennessee 37232-0146, USA.
authors.forename: Zhongmei
authors.initials: Z
authors.lastname: Tang
authors.forename: Martha V
authors.initials: MV
authors.lastname: Martin
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005227:Fatty Acids / Q000032:analysis / Q000378:metabolism; D006801:Humans; D006888:Hydroxycholesterols / Q000032:analysis / Q000378:metabolism; D007553:Isotope Labeling; D007700:Kinetics; D008110:Liver Extracts / Q000378:metabolism; D013058:Mass Spectrometry; D055432:Metabolomics / Q000379:methods*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D015203:Reproducibility of Results; D012984:Software; D013379:Substrate Specificity; D014020:Tissue Extracts / Q000378:metabolism*; D017186:Titrimetry
chemical_list: D005227:Fatty Acids; D006888:Hydroxycholesterols; D008110:Liver Extracts; D014020:Tissue Extracts; C011724:cholest-5-en-3 beta,7 alpha-diol; D003577:Cytochrome P-450 Enzyme System; D010100:Oxygen