Computational identification and binding analysis of orphan human cytochrome P450 4X1 enzyme with substrates
© Kumar; licensee BioMed Central. 2015
Received: 12 August 2014
Accepted: 5 January 2015
Published: 17 January 2015
Cytochrome P450s (CYPs) are important heme-containing proteins, well known for their monooxygenase reaction. The human cytochrome P450 4X1 (CYP4X1) is categorized as “orphan” CYP because of its unknown function. In recent studies it is found that this enzyme is expressed in neurovascular functions of the brain. Also, various studies have found the expression and activity of orphan human cytochrome P450 4X1 in cancer. It is found to be a potential drug target for cancer therapy. However, three-dimensional structure, the active site topology and substrate specificity of CYP4X1 remain unclear.
In the present study, the three-dimensional structure of orphan human cytochrome P450 4X1 was generated by homology modeling using Modeller 9v8. The generated structure was accessed for geometrical errors and energy stability using PROCHECK, VERFIY 3D and PROSA. A molecular docking analysis was carried out against substrates arachidonic acid and anandamide and the docked substrates were predicted for drug-likeness, ADME-Tox parameters and biological spectrum activity.
The three-dimensional model of orphan human cytochrome P450 4X1 was generated and assessed with various structural validation programmes. Docking of orphan human cytochrome P450 4X1 with arachidonic acid revealed that TYR 112, ALA 126, ILE 222, ILE 223, THR 312, LEU 315, ALA 316, ASP 319, THR 320, PHE 491 and ILE 492 residues were actively participating in the interaction, while docking of CYP4X1 with anandamide showed that TYR 112, GLN 114, PRO 118, ALA 126, ILE 222, ILE 223, SER 251, LEU 315, ALA 316 and PHE 491 key residues were involved in strong interaction.
From this study, several key residues were identified to be responsible for the binding of arachidonic acid and anandamide with orphan human cytochrome P450 4X1. Both substrates obeyed Lipinski rule of five in drug-likeness test and biological spectrum prediction showed anticarcinogenic activity. Compared to anandamide, arachidonic acid showed strong interaction with cytochrome P450 4X1 and also less health effect in certain human system in ADME-Tox prediction. These findings provide useful information on the biological role and structure-based drug design of orphan human cytochrome P450 4X1.
Cytochromes P450 are a super family of heme-thiolate proteins which are involved in the oxidative metabolism of both foreign and endogenous compounds . The P450 enzymes are divided into 18 families and 43 subfamilies. The human P450s can also be divided based on their ability to metabolize xenobiotic compounds: sterols, xenobiotics, fatty acids, eicosanoids, and vitamins, and the orphans . About 1/4 of the human P450 enzymes are considered to be “orphans” because, functional information, expression patterns, and regulation are still largely unknown. The orphan enzymes are mostly found in families 1–4, with the largest number found in the P450 4 family [3-5].
CYP4X1 (cytochrome P450, family 4, subfamily X, polypeptide 1) is a protein which in humans is encoded by CYP4X1 gene and is considered as one of such “orphan” CYPs. The human CYP4X1 gene is located within the P450 ABXZ gene cluster on chromosome 1. The gene has 12 exons and the predicted protein has 509 amino acids . The tissue distribution of human CYP4X1 is reported to be predominantly found in adult human skeletal muscle, trachea, and aorta . Recent report suggests that CYP4X1 is expressed in brain and in the liver [8,9]. It is involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. Members of the cytochrome P450 4F subfamily are known to primarily oxidize endogenous compounds, for example, fatty acids and arachidonic acid derivatives . The metabolic capabilities of CYP4X1 are largely unknown, yet a recent study has identified arachidonic acid derivatives to have been implicated in a large number of physiologically important processes. A number of P450s, primarily from subfamilies 2C, 2J, 4A and 4F, are known to oxidize arachidonic acid which has been implicated as important signaling mediator. The arachidonic acid derivative anandamide (arachidonoyl ethanol amide) is a natural endocannabinoid found in most human tissues, and acts as an important signaling mediator in neurological, immune and cardiovascular functions. Anandamide (arachidonoyl ethanol amide) has emerged as an important signaling molecule in the neurovascular cascade .
Human CYP4X1 amino acid sequence has been identified recently but the three-dimensional structure of this protein is not yet known. Earlier experimental studies of CYP4X1 proposed that arachidonic acid and its derivative anandamide can act as possible substrates . However, to date information on the structure and ligand binding site is not available for CYP4X1. Through homology modeling it is possible to generate realistic model comparable to experimental structures and through docking studies substrate binding energies and important key residues involved in substrate binding can be found. Many Computer-Aided Drug Design (CADD) methodologies have been carried out previously for finding suitable drug target [13-19]. In the present work, three-dimensional model of CYP4X1 was constructed using homology modeling and energy minimization was done to refine the model. After that, arachidonic acid and anandamide were docked into the active sites of the CYP4X1 model. The interaction between CYP4X1 and substrates helped in finding energetically favorable binding sites and the key residues responsible for substrate specificity.
The sequence of human CYP4X1 protein in FASTA format was retrieved from Uniprot Knowledge base (http://www.uniprot.org/) of accession number Q8N118.
Sequence alignment and homology modeling
For the template selection, PSI-BLAST search was used against Protein Data Bank (PDB) and top ranked six templates (1TQN, 3CZH, 2HI4, 3NA0, 3K9V, 3E4E) were selected for the model building. The templates and target sequence were aligned by using Clustal Omega  with default parameters and observed for conserved sequence. Further, the aligned sequence was used as the input to generate homology model of CYP4X1 using Modeller 9v5 . The coordinates for heme were obtained from the template 1TQN and positioned as in the template.
Energy minimization and structural validation
The constructed CYP4X1 model was further refined by energy minimization using YASARA package , and the resulting model was subjected to structural quality assessment. PROCHECK and VERIFY 3D were used for geometric evaluation. The PROSA program was used to assess the energy of residue-residue interaction using a distance-based pair potential and the energy was transformed to a score called Z-score. Residues with negative Z-score indicate reasonable side-chain interactions.
Binding site analysis
ConSurf was used for identification of the functional regions in the protein. The degree of conservation of the amino acid sites among homologues protein with similar sequences was estimated. The conservation scores were depicted onto the molecular surface of the orphan human cytochrome P450 4X1 to reveal the patches with highly conserved residues that are often important for biological function.
The possible substrates like arachidonic acid and its derivative anandamide were downloaded from the PubChem in Structure Data Format (SDF). Conversion of SDF to Protein Data Bank (PDB) format was carried out using Open Babel program . The MMFF94 force field was used for energy minimization of ligand molecules . Gasteiger partial charges were added to the ligand atoms, non-polar hydrogen atoms were merged and rotatable bonds were defined. Ligand geometries and electric properties were calculated using MOPAC2009 .
Docking calculations were carried out using DockingServer  to compute the free energy of binding on protein model. Essential hydrogen atoms, Kollman united atom type charges and solvation parameters were added with the aid of Auto Dock tools . Affinity (grid) maps of 60X60X60Å grid points and 0.375 Å spacing were generated using the Auto grid program. Auto Dock parameter set and distance dependent dielectric functions were used in the calculation of the Van der Waals and the electrostatic terms respectively. Docking simulations were performed using the Lamarckian Genetic Algorithm (LGA) and the Solis and Wets local search method. Initial position, orientation and torsions of the ligand molecules were set randomly and all rotatable torsions were released during docking. Each docking experiment was derived from 10 different runs that were set to terminate after a maximum of 250000 energy evaluations. During the search population size of 150, translational step of 0.2 Å and quaternion and torsion steps of 5 were applied.
Prediction of drug-likeness, ADME-Tox and biological spectrum activity
The substrates arachidonic acid and anandamide were subjected drug-likeness prediction using Lipinski rule of five, toxicity prediction using ADME-Tox and also biological activity prediction.
Results and discussion
Binding site prediction
ConSurf  was used to characterize the functional regions in the protein. It identifies by considering the degree of conservation of the amino acid sites among their sequence homologues. The conservation grades were projected on the molecular surface of the CYP4X1 protein to reveal the patches with highly conserved residues that are often important for biological function. The surface residues with the most conserved amino acids are shown colored in bordeaux, residues with average conservation in white and variable amino acids in turquoise in the protein structure in Figure 3B.
Predicting ligand binding sites can reduce the conformational space of docking and also can provide insights into their molecular functions.
Docking of CYP4X1 with selected ligands
Free energy of binding (kcal /mol)
Inhibition constant, Ki (uM)
vdW + Hbond + desolv energy (kcal /mol)
Electrostatic energy (kcal /mol)
Total inter molec. energy (kcal/ mol)
Details of intermolecular interactions in the binding site of orphan human cytochrome P450 4X1
O1 [3.81]- GLN114
C4 [3.87] - TYR112
C8 [3.80]- GLN114
C17 [2.99] - ILE223
C13 [3.35]- GLN114
C14 [3.51] - ILE223
C18 [3.77] - GLN114
C19 [3.80] - ILE223
C4 [3.83] - GLN114
C12 [3.29] - PHE247
O2 [3.49]- ALA126
C15 [3.89] - PHE247
O2 [3.53] - LEU315
C17 [3.40] - LEU315
C6 [3.78] - SER385
C14 [3.27]- LEU315
C10 [3.53] - LEU315
C11[3.43] - LEU315
C19 [3.41] - PHE491
C6 [3.40] - PHE491
C9 [3.70] - PHE491
C5 [3.70] - PHE491
C2 [3.38] – PHE491
C4 [3.51] – PHE491
C16 [3.25] -TYR112
C16 [3.86] -TYR112
C1 [3.43] -LEU121
C8 [3.70] -LEU121
C10 [3.89] -GLN114
C3 [3.89] - LEU121
C2 [3.81] -LEU121
C21 [3.24] -GLN114
C4 [3.43] -LEU121
C19 [3.09] -GLN114
C1 [3.78] -ALA126
O1 [3.24]- ALA126
C4 [3.48] -ALA126
O2 [3.36]- PHE491
C18 [3.80] - ILE222
C20 [3.76] - ILE222
C14 [3.73] -ILE223
C11 [3.84] - ILE223
C20 [3.56] - HIS225
C17 [3.66] - HIS225
C10 [3.75] - PHE247
C3 [3.54]- LEU315
C5 [3.83] -ALA316
C7 [3.29] - ALA316
C19 [3.34] -PHE491
C21 [3.48] -PHE491
C14 [3.51] -PHE491
C11 [3.70] - PHE491
C15 [3.85] -PHE491
C22 [3.76] -PHE491
Evaluation of drug-likeness and ADME-Tox
Solubilitya H2O (mg mL−1)
LogDb pH 1.7 (stomach)
LogDb pH 4.6 (duodenum)
LogDb pH 6.5 (jejunum, ileum)
LogDb pH 7.4 (blood)
LogDb pH 8.0 (colon)
% oral bioavailabilityc
Absorptiond (cm s−1)
Distributione (L kg−1)
Prob. of Blood effectg
Prob. of Cardiovascular system effectg
Prob. of gastrointestinal system effectg
Prob. of kidney effectg
Prob. of liver effectg
Prob. of lung effectg
Biological spectrum predictions
Computer software PASS [38,39] predicts simultaneously several hundreds of biological activities depending upon the chemical structures of compounds such as the predicted activity spectrum giving probable activity (Pa) and probable inactivity (Pi). Prediction of this spectrum by PASS is based on SAR analysis of the training set containing more than 35,000 compounds, which correlates with more than 500 kinds of biological activities. Pa and Pi values are independent and their values can vary from 0 to 1.
Biological spectrum predictions of arachidonic acid and anandamide
Urologic disorders treatment
Antineoplastic (non-small cell lung cancer)
Digestive functional disorders treatment
Antineoplastic (gastric cancer)
Antineoplastic (head/neck cancer)
Allergic conjunctivitis treatment
The orphan human cytochrome P450 4X1 has been suggested to be a potential drug target for cancer therapy. Lack of the structural information about this enzyme hinders the detailed characterization of its biological functions and its applications in structure based design. For this reason, the three-dimensional model of orphan human cytochrome P450 4X1 was constructed using homology modeling. To provide useful information to characterize the enzyme’s function, two known substrates, arachidonic acid and anandamide were docked into the active sites and then refined by energy minimization to determine favorable binding modes. Several key residues TYR 112, ILE 223, LEU 315, ALA 316 and PHE 491 were identified to have involved in binding of arachidonic acid and anandamide. These key residues are expected to affect the catalytic activity and can be used as candidates for further mutagenesis studies. Both the substrates does not violate Lipinski rule of five in drug-likeness test, while in ADME-Tox prediction, arachidonic acid shows less health effects on cardiovascular system, gastrointestinal system and kidney of human when compared to anandamide. In biological activity spectrum predictions, both arachidonic acid and anandamide show anticarcinogenic activity. However, further in-vivo validation and conformation of the present finding is required. The results of this study will be useful for structure-based drug design of orphan human cytochrome P450 4X1.
I would like to acknowledge the computing facilities provided by Centre for Bioinformatics Research, Institute of Systems Biology (INBIOSIS), University Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia.
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