Oxidation of endogenous N-arachidonoylserotonin by human cytochrome P450 2U1

Abstract

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.

Keywords: Arachidonic Acid; Cytochrome P450; Eicosanoid; Lipid Oxidation; Mass Spectrometry (MS).

FIGURE 1.

Structures of N-arachidonoylserotonin and 2-oxo-N-arachidonoylserotonin.

FIGURE 2.

Reduced ferrous-CO versus ferrous difference spectrum of purified recombinant P450 2U1. The calculated P450 concentration was 3.0 μm (32).

FIGURE 3.

P450 2U1 N-terminal peptides identified by Edman degradation. A, preparative SDS-polyacrylamide gel of P450 2U1 protein. Electrophoresis consistently showed two protein bands, marked with arrows on the right side (direction of migration, top to bottom). Colloidal Coomassie Blue staining (1 μg of protein applied to gel) was used, and the indicated bands were excised and submitted to proteomic analysis. B, repetitive yields of amino acids recovered in Edman degradation cycles. The yields were fit to semilogarithmic plots. C, assignments. Residues 2–10 are from the upper SDS gel band. Peptides 34–44, 36–46, and 43–53 are from the lower SDS gel band.

FIGURE 4.

Proteomic analysis of upper and lower protein bands of the P450 2U1. The yellow shading shows the peptides that were identified in each protein band (Fig. 3A). The Cys and Met residues are shaded green to indicate that these residues were purposely oxidized in the protein workup. A, from upper band. B, from lower band.

FIGURE 5.

HPLC radiochromatogram for oxidation of [1-¹⁴C]arachidonic acid by P450 2U1. The ω- and ω-1 hydroxylation products and the residual substrate are labeled. The nature of the radioactivity eluting at the void volume is unknown.

FIGURE 6.

LC-MS chromatograms from a bovine brain doublet search experiment. A, sample derived from brain tissue extract, NADPH-generating system, and inactivated P450 2U1 enzyme. B, sample generated with P450 2U1 incubated in an ¹⁶O₂ atmosphere. C, sample from two reaction mixtures, incubated either with ¹⁶O₂ or ¹⁸O₂, combined together. m/z 479 (1-O atom addition to N-arachidonoylserotonin) was monitored in each case.

FIGURE 7.

2-Oxygenation of N-arachidonoylserotonin by P450 2U1. A, minus P450 2U1. B, plus P450 2U1. The scales are identical, and the t_R of 2-oxo-N-arachidonoylserotonin is marked with the double-headed red arrow. The m/z 479 peak in the blank reaction (A) appeared at the position of N-arachidonoylserotonin and is attributed to a solvent adduct (+16), as revealed by fragmentation. The peak eluted at t_R 16.8 min in B was not identified.

FIGURE 8.

Binding of N-arachidonoylserotonin to P450 2U1. Each of two 1-ml cuvettes contained 3.0 μm P450 2U1. A baseline was recorded, and 6 μm N-arachidonoylserotonin was added to the sample cuvette, prior to rescanning.

FIGURE 9.

LC-MS (APCI⁺) fragmentation patterns of N-arachidonoylserotonin products formed in the presence of either P450 2U1 or P450 2W1 and comparison with the fragmentation spectrum of the substrate. The fragment with m/z 193 shows the presence of an additional oxygen moiety in the indole moiety of N-arachidonoylserotonin. A, P450 2U1 reaction product; B, P450 2W1 reaction product; C, substrate.

FIGURE 10.

¹H NMR spectrum of the aromatic region (δ 6–8 ppm) of the HPLC-purified product from a P450 2U1 incubation with N-arachidonoylserotonin. Proton numbering of the indole ring is indicated. A, one-dimensional spectrum. B, two-dimensional COSY spectrum.

FIGURE 11.

Mass fragmentation patterns A, synthetic 2-oxo-N-arachidonoylserotonin B, product of oxidation of N-arachidonoylserotonin by P450 2U1. An APCI⁺ source was used.

FIGURE 12.

Steady-state kinetic analysis of oxidation of N-arachidonoylserotonin to 2-oxo-N-arachidonoylserotonin by P450 2U1. k_cat 32 (± 9) pmol product formed min⁻¹ (nmol P450 2U1)⁻¹; K_m = 82 ± 2 μm.

FIGURE 13.

Inhibition of fatty acid amide hydrolase by synthetic N-arachidonoylserotonin and 2-oxo-N-arachidonoylserotonin. The IC₅₀ for N-arachidonoylserotonin was 14 ± 1 μm. The IC₅₀ for 2-oxo-N-arachidonoylserotonin was 47 ± 5 μm.