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 specifi city, the purifi ed 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 identifi ed 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 (9 R ,10 S ) over (9 S ,10 R ). Although the genomic sequences of human and numerous other organisms have been established, the functions of less than one-half of the proteins have been annotated, even in Escherichia coli . Thus, an important and challenging

Colon Cancer–Specific Cytochrome P450 2W1 Converts Duocarmycin Analogues into Potent Tumor Cytotoxins

“…Clinical studies indicate that increased levels of CYP2W1 expression positively correlate with the degree of tumor malignancy, as well as with decreased 10-year survival in patients with colon cancer (12,13). So far, several groups have reported on the metabolism of a few candidate substrates (i.e., benzopyrene, sterigmatocystin, aflatoxin B1, lysophospholipids, and fluorobenzothiazoles) for CYP2W1 expressed in E. coli or mammalian cells (14)(15)(16)(17)(18)(19). However, the potential of CYP2W1 as a specific target in cancer chemotherapy remains hitherto unexplored.…”

Oxidation of Endogenous N-Arachidonoylserotonin by Human Cytochrome P450 2U1

“…Fatty acids are known to be hydroxylated by P450s at the -carbon and the -1, -2, and -3 positions (3, 51, 52). However, midchain hydroxylations (14) and ␣-and ␤-hydroxylations (53,54) are also known. We found that P450 2U1 yielded a reverse type I difference spectrum (46), generally considered to indicate a change in the iron spin state (from high to low spin), because of an increase in the occupancy of H 2 O as the distal ligand.…”

“…Other metabolomics data mining software was also used, e.g. MZmine (39) and an in-house Matlab program (14) for doublet searches (M, M ϩ 2) in the case of 18 O-based isotopic labeling experiments. MZmine2 (40) serves as a high performance tool in differential analysis of data from multiple LC-MS experiments, enabling noise filtering, peak detection, and retention time (t R ) alignment for corresponding peaks from different runs (40).…”

“…The peak list was copied to an Excel file and exported as a CSV (comma separated values) file. An in-house Matlab program was used to extract peak information from the CSV file and in searches for 18 O/ 16 O doublet patterns (14).…”

“…Still another approach is a "metabolomics" one, involving an "unbiased" search in an extract of a tissue in which the enzyme is normally found (12,13). Some results with the human P450s have been compiled (14,15). It is also possible that a particular P450 may not have true endogenous substrates, e.g.…”