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

Background: Cytochrome P450 (P450, CYP) 2U1 is an orphan enzyme, only reported to catalyze some fatty acid oxidations. Results: Metabolomic screening led to the identification of N-arachidonoylserotonin as a substrate. Conclusion: P450 2U1 catalyzes oxygenation at C-2 of the indole ring. Significance: The localization of P450 2U1 and its metabolism of an inhibitor of fatty acid amide hydrolase may indicate a function in brain. Cytochrome P450 (P450) 2U1 has been shown to be expressed, at the mRNA level, in human thymus, brain, and several other tissues. Recombinant P450 2U1 was purified and used as a reagent in a metabolomic search for substrates in bovine brain. In addition to fatty acid oxidation reactions, an oxidation of endogenous N-arachidonoylserotonin was characterized. Subsequent NMR and mass spectrometry and chemical synthesis showed that the main product was the result of C-2 oxidation of the indole ring, in contrast to other human P450s that generated different products. N-Arachidonoylserotonin, first synthesized chemically and described as an inhibitor of fatty acid amide hydrolase, had previously been found in porcine and mouse intestine; we demonstrated its presence in bovine and human brain samples. The product (2-oxo) was 4-fold less active than N-arachidonoylserotonin in inhibiting fatty acid amide hydrolase. The rate of oxidation of N-arachidonoylserotonin was similar to that of arachidonic acid, one of the previously identified fatty acid substrates of P450 2U1. The demonstration of the oxidation of N-arachidonoylserotonin by P450 2U1 suggests a possible role in human brain and possibly other sites.