Functional Studies of Orphan Enzymes by LC‐MS Based Metabolomics: Oxidation of Endogenous N‐Arachidonoylserotonin by Human Cytochrome P450 2U1 (LB174)

Abstract

Cytochrome P450 (P450) 2U1 has been shown to be expressed (at the mRNA level) in human brain and thymus. In order to elucidate the physiological function of this enzyme, an LC‐MS‐based metabolomics and isotopic labeling approach was used to identify endogenous substrate of human P450 2U1. In addition to fatty acid oxidation reactions, oxidation of endogenous N‐arachidonoylserotonin was characterized. Subsequent NMR, mass spectrometry, and comparison with a synthesized chemical standard showed that the main product was the result of C2 oxidation of the indole ring, in contrast to other P450s that generated different products. The product (2‐oxo) was 4‐fold less active than N‐arachidonoylserotonin in inhibiting fatty acid amide hydrolase. The oxidation rate of N‐arachidonoylserotonin was similar to that of arachidonic acid, one of the previously identified fatty acid substrates of P450 2U1. The presence of N‐arachidonoylserotonin in human brain and the oxidation of N‐arachidonoylserotonin by P450 2U1 suggest a possible role in human brain and possibly other sites. (Supported in part by USPHS R37 CA090426)