Spectral and 3D model studies of the interaction of orphan human cytochrome P450 2U1 with substrates and ligands

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 1; pp. 3144 - 3153
• Main Authors: Dhers, Laura, Pietrancosta, Nicolas, Ducassou, Lionel, Ramassamy, Booma, Dairou, Julien, Jaouen, Maryse, André, François, Mansuy, Daniel, Boucher, Jean-Luc
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2017; Elsevier
• Subjects: active sites; arachidonic acid; Arachidonic Acid - chemistry; Arachidonic Acid - metabolism; Arachidonic acid derivatives; Arachidonic Acids - chemistry; Arachidonic Acids - metabolism; Biocatalysis; cytochrome P-450; Cytochrome P450 Family 2 - chemistry; Cytochrome P450 Family 2 - metabolism; Debrisoquin - chemistry; electron paramagnetic resonance spectroscopy; Electron Spin Resonance Spectroscopy; Escherichia coli; Expression in E. coli; genome; Humans; Imidazole ligands; Imidazoles - chemistry; ketoconazole; Lauric Acids - chemistry; Life Sciences; Ligands; miconazole; Models, Molecular; Molecular docking; Molecular Docking Simulation; molecular models; Orphan cytochrome P450 2U1; Oxidation-Reduction; Protein Binding; pyridines; Pyridines - chemistry; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Serotonin - analogs & derivatives; Serotonin - chemistry; Serotonin - metabolism; spectral analysis; Spectrophotometry, Ultraviolet; Substrate Specificity; xenobiotics; AA; Deb; Im; Arachidonic acid derivatives; Terfimidazole; Molecular docking; BSA; AS; Orphan cytochrome P450 2U1; LA; IPTG; CYP; DLPC; Imidazole ligands; Expression in E. coli; B3S; LSOD
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2016.07.018

Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. It is considered as an “orphan” protein as few data are available on its physiological function(s) and spectral characteristics. Its only known substrates reported so far are unsaturated fatty acids such as arachidonic acid (AA), and, more recently, N-arachidonoylserotonin (AS) and some xenobiotics related to debrisoquine (Deb) and terfenadine. We have expressed CYP2U1 in E. coli and performed UV–vis and EPR spectroscopy experiments with purified CYP2U1 alone and in the presence of substrates and imidazole and pyridine derivatives. Docking experiments using a 3D homology model of CYP2U1 were done to explain the observed spectroscopic data and the different regioselectivities of the oxidations of AA and AS. The UV–vis and EPR spectra of native recombinant human CYP2U1 revealed a predominant low-spin hexacoordinate FeIII state. Imidazole (Im) derivatives, such as miconazole, acted as FeIII ligands, contrary to ketoconazole, whereas the previously described substrates AS and Deb led to “reverse type I” difference UV–vis spectra. These data, as well as the different regioselectivities of AA and AS oxidations, were supported by docking experiments performed on our previously reported CYP2U1 3D model. Our study describes for the first time the mode of interaction of several FeIII-heme ligands and substrates with the active site of CYP2U1 on the basis of spectroscopic and molecular docking data. The good agreement between these data validates the used CYP2U1 3D model which should help the design of new substrates or inhibitors of this orphan CYP. •Few data are available on orphan human cytochrome P450 2U1.•N-Ter truncated, soluble, human CYP2U1 was expressed in E. coli.•UV–vis and EPR spectroscopy binding experiments of substrates and imidazoles were performed.•Docking experiments in our 3D model explained the spectroscopic data.