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, 275 (14), 3706-17

Expression and Purification of Orphan Cytochrome P450 4X1 and Oxidation of Anandamide

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Expression and Purification of Orphan Cytochrome P450 4X1 and Oxidation of Anandamide

Katarina Stark et al. FEBS J.

Abstract

Cytochrome P450 (P450) 4X1 is one of the so-called 'orphan' P450s without an assigned biological function. Codon-optimized P450 4X1 and a number of N-terminal modified sequences were expressed in Escherichia coli. Native P450 4X1 showed a characteristic P450 spectrum but low expression in E. coli DH5alpha cells (< 100 nmol P450.L(-1)). The highest level of expression (300-450 nmol P450.L(-1) culture) was achieved with a bicistronic P450 4X1 construct (N-terminal MAKKTSSKGKL, change of E2A, amino acids 3-44 truncated). Anandamide (arachidonoyl ethanolamide) has emerged as an important signaling molecule in the neurovascular cascade. Recombinant P450 4X1 protein, co-expressed with human NADPH-P450 reductase in E. coli, was found to convert the natural endocannabinoid anandamide to a single monooxygenated product, 14,15-epoxyeicosatrienoic (EET) ethanolamide. A stable anandamide analog (CD-25) was also converted to a monooxygenated product. Arachidonic acid was oxidized more slowly to 14,15- and 8,9-EETs but only in the presence of cytochrome b(5). Other fatty acids were investigated as putative substrates but showed only little or minor oxidation. Real-time PCR analysis demonstrated extrahepatic mRNA expression, including several human brain structures (cerebellum, amygdala and basal ganglia), in addition to expression in human heart, liver, prostate and breast. The highest mRNA expression levels were detected in amygdala and skin. The ability of P450 4X1 to generate anandamide derivatives and the mRNA distribution pattern suggest a potential role for P450 4X1 in anandamide signaling in the brain.

Figures

Fig. 1
Fig. 1
Optimizations introduced into the P450 4X1 cDNA for E. coli expression. Upper line; predicted amino acid sequence; middle line, nucleotide sequence predicted from genomic sequence; lower line, nucleotide sequence optimized for E. coli expression.
Fig. 2
Fig. 2
N-Terminal modifications used for heterologous expression of P450 4X1 membranes in E. coli [18] (see also Supplementary material Fig. S2). Amino acid changes are in italics and underlined.
Fig. 3
Fig. 3
Fe2+-CO vs. Fe2+ difference spectra. A, P450 4X1 construct 3 expression was done in E. coli (with pGroES/EL12). The spectrum was recorded using 1/2 dilutions of whole cell extracts and reducing with Na2S2O4. B, Solubilized P450 4X1 (1.5 μM) C, Difference spectrum of purified P450 4X1 (0.14 μM).
Fig 4
Fig 4
SDS-polyacrylamide gel electrophoresis of purified recombinant P450 4X1. Lane 1, Mr markers; lane 2, purified P450 4X1 (4 pmol).
Fig. 5
Fig. 5
Tissue distribution of P450 4X1 mRNA measured by real-time PCR. The relative levels of P450 4X1 mRNA were determined using real-time PCR in the tissues indicated below, using GAPDH as a reference standard. Different human cDNAs were used as templates, and SYBR Green was used for detection. The mRNA levels are shown as the ratio of P450 4X1 to GAPDH and represent the mean of triplicate measurements from each sample. The relative expression was calculated using the ΔCt method (Livak). For graphic presentation the graphs have standard deviations shown.

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