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1.  Identification, Modeling and Ligand Affinity of Early Deuterostome CYP51s, and Functional Characterization of Recombinant Zebrafish Sterol 14α-Demethylase 
Biochimica et biophysica acta  2013;1840(6):1825-1836.
Background
Sterol 14α-demethylase (cytochrome P450 51, CYP51, P45014DM) is a microsomal enzyme that in eukaryotes catalyzes formation of sterols essential for cell membrane function and as precursors in biosynthesis of steroid hormones. Functional properties of CYP51s are unknown in non-mammalian deuterostomes.
Methods
PCR-cloning and sequencing and computational analyses (homology modeling and docking) addressed CYP51 in zebrafish Danio rerio, the reef fish sergeant major Abudefduf saxatilis, and the sea urchin Strongylocentrotus purpuratus. Following N-terminal amino acid modification, zebrafish CYP51 was expressed in Escherichia coli, and lanosterol 14α-demethylase activity and azole inhibition of CYP51 activity were characterized using GC/MS.
Results
Molecular phylogeny positioned S. purpuratus CYP51 at the base of the deuterostome clade. In zebrafish, CYP51 is expressed in all organs examined, most strongly in intestine. The recombinant protein bound lanosterol and catalyzed 14α-demethylase activity, at 3.2 nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave KS (dissociation constant) values of 0.26 μM for ketoconazole and 0.64 μM for propiconazole. Displacement of carbon monoxide also indicated zebrafish CYP51 has greater affinity for ketoconazole. Docking to homology models showed that lanosterol docks in fish and sea urchin CYP51s with an orientation essentially the same as in mammalian CYP51. Docking of ketoconazole indicates it would inhibit fish and sea urchin CYP51s.
Conclusions
Biochemical and computational analyses are consistent with lanosterol being a substrate for early deuterostome CYP51s.
General Significance
The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications.
doi:10.1016/j.bbagen.2013.12.009
PMCID: PMC4118742  PMID: 24361620
Sterol 14α-demethylase; CYP51; zebrafish; deuterostomes; sterol biosynthesis; azole inhibition
2.  CYB5D2 Requires Heme-Binding to Regulate HeLa Cell Growth and Confer Survival from Chemotherapeutic Agents 
PLoS ONE  2014;9(1):e86435.
The cytochrome b5 domain containing 2 (CYB5D2; Neuferricin) protein has been reported to bind heme, however, the critical residues responsible for heme-binding are undefined. Furthermore, the relationship between heme-binding and CYB5D2-mediated intracellular functions remains unknown. Previous studies examining heme-binding in two cytochrome b5 heme-binding domain-containing proteins, damage-associated protein 1 (Dap1; Saccharomyces cerevisiae) and human progesterone receptor membrane component 1 (PGRMC1), have revealed that conserved tyrosine (Y) 73, Y79, aspartic acid (D) 86, and Y127 residues present in human CYB5D2 may be involved in heme-binding. CYB5D2 binds to type b heme, however, only the substitution of glycine (G) at D86 (D86G) within its cytochrome b5 heme-binding (cyt-b5) domain abolished its heme-binding ability. Both CYB5D2 and CYB5D2(D86G) localize to the endoplasmic reticulum. Ectopic CYB5D2 expression inhibited cell proliferation and anchorage-independent colony growth of HeLa cells. Conversely, CYB5D2 knockdown and ectopic CYB5D2(D86G) expression increased cell proliferation and colony growth. As PGRMC1 has been reported to regulate the expression and activities of cytochrome P450 proteins (CYPs), we examined the role of CYB5D2 in regulating the activities of CYPs involved in sterol synthesis (CYP51A1) and drug metabolism (CYP3A4). CYB5D2 co-localizes with cytochrome P450 reductase (CYPOR), while CYB5D2 knockdown reduced lanosterol demethylase (CYP51A1) levels and rendered HeLa cells sensitive to mevalonate. Additionally, knockdown of CYB5D2 reduced CYP3A4 activity. Lastly, CYB5D2 expression conferred HeLa cell survival from chemotherapeutic agents (paclitaxel, cisplatin and doxorubicin), with its ability to promote survival being dependent on its heme-binding ability. Taken together, this study provides evidence that heme-binding is critical for CYB5D2 in regulating HeLa cell growth and survival, with endogenous CYB5D2 being required to modulate CYP activities.
doi:10.1371/journal.pone.0086435
PMCID: PMC3899279  PMID: 24466094
3.  Functional Analysis of the Unique Cytochrome P450 of the Liver Fluke Opisthorchis felineus 
PLoS Neglected Tropical Diseases  2015;9(12):e0004258.
The basic metabolic cytochrome P450 (CYP) system is essential for biotransformation of sterols and xenobiotics including drugs, for synthesis and degradation of signaling molecules in all living organisms. Most eukaryotes including free-living flatworms have numerous paralogues of the CYP gene encoding heme monooxygenases with specific substrate range. Notably, by contrast, the parasitic flatworms have only one CYP gene. The role of this enzyme in the physiology and biochemistry of helminths is not known. The flukes and tapeworms are the etiologic agents of major neglected tropical diseases of humanity. Three helminth infections (Opisthorchis viverrini, Clonorchis sinensis and Schistosoma haematobium) are considered by the International Agency for Research on Cancer (IARC) as definite causes of cancer. We focused our research on the human liver fluke Opisthorchis felineus, an emerging source of biliary tract disease including bile duct cancer in Russia and central Europe. The aims of this study were (i) to determine the significance of the CYP activity for the morphology and survival of the liver fluke, (ii) to assess CYP ability to metabolize xenobiotics, and (iii) to localize the CYP activity in O. felineus tissues. We observed high constitutive expression of CYP mRNA (Real-time PCR) in O. felineus. This enzyme metabolized xenobiotics selective for mammalian CYP2E1, CYP2B, CYP3A, but not CYP1A, as determined by liquid chromatography and imaging analyses. Tissue localization studies revealed the CYP activity in excretory channels, while suppression of CYP mRNA by RNA interference was accompanied by morphological changes of the excretory system and increased mortality rates of the worms. These results suggest that the CYP function is linked to worm metabolism and detoxification. The findings also suggest that the CYP enzyme is involved in vitally important processes in the organism of parasites and is a potential drug target.
Author Summary
The basic metabolic system CYP (cytochrome P450) is essential for biotransformation of sterols and xenobiotics, for synthesis and degradation of signaling molecules in all living organisms. Most eukaryotes including free-living flatworms evolved numerous paralogues of the CYP gene. Notably, by contrast, flukes and tapeworms–the etiologic agents of major neglected tropical diseases of humanity, have only one gene. However, the role of P450 in the physiology and biochemistry of helminths is not known. This report presents the first functional study of the CYP enzyme of any of the parasitic flatworms. We focused our research on the food-borne human liver fluke, Opisthorchis felineus, an emerging source of biliary tract diseases in Russia, Kazakhstan and central Europe. Here we report that this liver fluke has evolved a highly expressed functional monooxygenase system with broad substrate specificity. Tissue localization studies and suppression of CYP mRNA by RNA interference revealed the CYP function is linked to the excretory system and possibly to metabolism and detoxification. The fluke’s monooxygenase likely is a model for orthologues of the singular CYP of parasitic flatworms at large, where it plays a critical role in the pathogen’s metabolism that contributes to worm survival and drug resistance.
doi:10.1371/journal.pntd.0004258
PMCID: PMC4666407  PMID: 26625139
4.  Whole-Genome Identification, Phylogeny, and Evolution of the Cytochrome P450 Family 2 (CYP2) Subfamilies in Birds 
Genome Biology and Evolution  2016;8(4):1115-1131.
The cytochrome P450 (CYP) superfamily defends organisms from endogenous and noxious environmental compounds, and thus is crucial for survival. However, beyond mammals the molecular evolution of CYP2 subfamilies is poorly understood. Here, we characterized the CYP2 family across 48 avian whole genomes representing all major extant bird clades. Overall, 12 CYP2 subfamilies were identified, including the first description of the CYP2F, CYP2G, and several CYP2AF genes in avian genomes. Some of the CYP2 genes previously described as being lineage-specific, such as CYP2K and CYP2W, are ubiquitous to all avian groups. Furthermore, we identified a large number of CYP2J copies, which have been associated previously with water reabsorption. We detected positive selection in the avian CYP2C, CYP2D, CYP2H, CYP2J, CYP2K, and CYP2AC subfamilies. Moreover, we identified new substrate recognition sites (SRS0, SRS2_SRS3, and SRS3.1) and heme binding areas that influence CYP2 structure and function of functional importance as under significant positive selection. Some of the positively selected sites in avian CYP2D are located within the same SRS1 region that was previously linked with the metabolism of plant toxins. Additionally, we find that selective constraint variations in some avian CYP2 subfamilies are consistently associated with different feeding habits (CYP2H and CYP2J), habitats (CYP2D, CYP2H, CYP2J, and CYP2K), and migratory behaviors (CYP2D, CYP2H, and CYP2J). Overall, our findings indicate that there has been active enzyme site selection on CYP2 subfamilies and differential selection associated with different life history traits among birds.
doi:10.1093/gbe/evw041
PMCID: PMC4860681  PMID: 26979796
avian genomes; cytochrome P450 (CYPs); substrate recognition sites (SRS); heme binding areas (HEM); positive selection
5.  Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae) 
Journal of Insect Science  2015;15(1):122.
Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system.
doi:10.1093/jisesa/iev100
PMCID: PMC4672210  PMID: 26320261
Spodoptera exigua; cytochrome P450; CYP9A11; CPR; expression
6.  CYP51 structures and structure-based development of novel, pathogen-specific inhibitory scaffolds 
Graphical abstract
Highlights
► CYP51s (sterol 14alpha-demethylases) are efficient drug target enzymes. ► CYP51s have a highly rigid substrate binding cavity. ► CYP51 structure-based development of a new inhibitory scaffold is described.
CYP51 (sterol 14α-demethylase) is a cytochrome P450 enzyme essential for sterol biosynthesis and the primary target for clinical and agricultural antifungal azoles. The azoles that are currently in clinical use for systemic fungal infections represent modifications of two basic scaffolds, ketoconazole and fluconazole, all of them being selected based on their antiparasitic activity in cellular experiments. By studying direct inhibition of CYP51 activity across phylogeny including human pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum, we identified three novel protozoa-specific inhibitory scaffolds, their inhibitory potency correlating well with antiprotozoan activity. VNI scaffold (carboxamide containing β-phenyl-imidazoles) is the most promising among them: killing T. cruzi amastigotes at low nanomolar concentration, it is also easy to synthesize and nontoxic. Oral administration of VNI (up to 400 mg/kg) neither leads to mortality nor reveals significant side effects up to 48 h post treatment using an experimental mouse model of acute toxicity. Trypanosomatidae CYP51 crystal structures determined in the ligand-free state and complexed with several azole inhibitors as well as a substrate analog revealed high rigidity of the CYP51 substrate binding cavity, which must be essential for the enzyme strict substrate specificity and functional conservation. Explaining profound potency of the VNI inhibitory scaffold, the structures also outline guidelines for its further development. First steps of the VNI scaffold optimization have been undertaken; the results presented here support the notion that CYP51 structure-based rational design of more efficient, pathogen-specific inhibitors represents a highly promising direction.
doi:10.1016/j.ijpddr.2012.06.001
PMCID: PMC3596085  PMID: 23504044
Sterol 14α-demethylase; CYP51; Inhibition; Crystal structure
7.  Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata 
Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell.
PMCID: PMC89351  PMID: 10390230
8.  Cytochrome P450 Monooxygenase CYP53 Family in Fungi: Comparative Structural and Evolutionary Analysis and Its Role as a Common Alternative Anti-Fungal Drug Target 
PLoS ONE  2014;9(9):e107209.
Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family's role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative structural (gene and protein structure-level) and evolutionary analysis of a fungal P450 family.
doi:10.1371/journal.pone.0107209
PMCID: PMC4164535  PMID: 25222113
9.  Genetic and structural analyses of cytochrome P450 hydroxylases in sex hormone biosynthesis: Sequential origin and subsequent coevolution 
Molecular phylogenetics and evolution  2015;94(Pt B):676-687.
Biosynthesis of steroid hormones in vertebrates involves three cytochrome P450 hydroxylases, CYP11A1, CYP17A1 and CYP19A1, which catalyze sequential steps in steroidogenesis. These enzymes are conserved in the vertebrates, but their origin and existence in other chordate subphyla (Tunicata and Cephalochordata) have not been clearly established. In this study, selected protein sequences of CYP11A1, CYP17A1 and CYP19A1 were compiled and analyzed using multiple sequence alignment and phylogenetic analysis. Our analyses show that cephalochordates have sequences orthologous to vertebrate CYP11A1, CYP17A1 or CYP19A1, and that echinoderms and hemichordates possess CYP11-like but not CYP19 genes. While the cephalochordate sequences have low identity with the vertebrate sequences, reflecting evolutionary distance, the data show apparent origin of CYP11 prior to the evolution of CYP19 and possibly CYP17, thus indicating a sequential origin of these functionally related steroidogenic CYPs. Co-occurrence of the three CYPs in early chordates suggests that the three genes may have coevolved thereafter, and that functional conservation should be reflected in functionally important residues in the proteins. CYP19A1 has the largest number of conserved residues while CYP11A1 sequences are less conserved. Structural analyses of human CYP11A1, CYP17A1 and CYP19A1 show that critical substrate binding site residues are highly conserved in each enzyme family. The results emphasize that the steroidogenic pathways producing glucocorticoids and reproductive steroids are several hundred million years old and that the catalytic structural elements of the enzymes have been conserved over the same period of time. Analysis of these elements may help to identify when precursor functions linked to these enzymes first arose.
Graphical abstract
doi:10.1016/j.ympev.2015.09.012
PMCID: PMC4801120  PMID: 26432395
cytochrome P450; steroid biosynthesis; pregnenolone; estrogen; progesterone; enzyme evolution
10.  Specific Effects of Potassium Ion Binding on Wild-Type and L358P Cytochrome P450cama 
Biochemistry  2006;45(48):14379-14388.
The camphor monoxygenase cytochrome P450cam (CYP101) requires potassium ion (K+) to drive formation of the characteristic high-spin state of the heme Fe+3 upon substrate binding. Amide 1H, 15N correlations in perdeuterated [U-15N] CYP101 were monitored as a function of K+ concentration by 2D-TROSY-HSQC in both camphor-bound oxidized (CYP-S) and camphor-and CO-bound reduced CYP101 (CYP-S-CO). In both forms, K+-induced spectral perturbations are detected in the vicinity of the K+ binding site proposed from crystallographic structures, but are larger and more widespread structurally in CYP-S than in CYP-S-CO. In CYP-S-CO, K+-induced perturbations occur primarily near the proposed K+ binding site in the B-B’ loop and B’ helix, which are also perturbed by binding of effector, putidaredoxin (Pdx). The spectral effects of K+ binding in CYP-S-CO oppose those observed upon Pdxr titration. However, Pdxr titration of CYP-S-CO in the absence of K+ results in multiple conformations. The spin-state equilibrium in the L358P mutant of CYP101 is more sensitive to K+ concentration than WT CYP101, consistent with a hypothesis that L358P preferentially populates conformations enforced by Pdx binding in WT CYP101. Thallium (I), a K+ mimic, minimizes the effects of Pdx titration on the NMR spectrum of CYP-S-CO, but is competent to replace K+ in driving the formation of high-spin CYP-S. These observations suggest that the role of K+ is to stabilize conformers of CYP-S that drive the spin state change prior to the first electron transfer, and that K+ stabilizes the CYP-S-CO conformer that interacts with Pdx. However, upon binding of Pdx, further conformational changes occur that disfavor K+ binding.
doi:10.1021/bi0617355
PMCID: PMC1764623  PMID: 17128977
CYP101; putidaredoxin; NMR; HSQC; electron transfer complex; TROSY; thallium; CYP101, cytochrome P450cam; CYP-S, oxidized camphor-bound CYP101; CYP-S-CO, reduced camphor- and carbonmonoxy-bound CYP101; IPTGm, isopropyl β-D-thiogalactoside; LB, Luria-Bertani media; NMR, nuclear magnetic resonance; HSQC, heteronuclear single-quantum correlation; M9, minimal growth medium; OD600, optical density at 600 nm; PdR, putidaredoxin reductase; Pdx, putidaredoxin; Pdxr, reduced putidaredoxin; TROSY, transverse relaxation optimized spectroscopy
11.  Identification of Two Different 14-α Sterol Demethylase-Related Genes (cyp51A and cyp51B) in Aspergillus fumigatus and Other Aspergillus species 
Journal of Clinical Microbiology  2001;39(7):2431-2438.
Two cyp51-related genes (cyp51A and cyp51B) encoding 14-α sterol demethylase-like enzymes were identified in the opportunistic human pathogen Aspergillus fumigatus. PCR amplification using degenerate oligonucleotides based on conserved areas of cytochrome P450 demethylases of other filamentous fungi and yeasts allowed the cloning and sequencing of two different homologue genes in A. fumigatus. Southern analysis confirmed that both genes hybridized to distinct genomic loci and that both are represented as single copies in the genome. Comparison of the deduced Cyp51A and Cyp51B proteins with the CYP51 proteins from Penicillium italicum, Aspergillus nidulans, Erysiphe graminis, Uncinula necator, Botrytis cinerea, Ustilago maydis, Cryptococcus neoformans, Candida albicans, Saccharomyces cerevisiae, Candida tropicalis, and Candida glabrata showed that the percentages of identity of the amino acid sequences (range, 40 to 70%) were high enough to consider Cyp51A and Cyp51B to be members of the fungal CYP51 family. Fragments from both genes were also cloned from other Aspergillus spp. (A. flavus, A. nidulans, and A. terreus). Phylogenetic analysis showed that, at least in the most pathogenic species of Aspergillus, there are two fungal CYP51 proteins. This is the first report of the existence of two homologue genes coding for 14-α sterol demethylase in the fungal kingdom. This finding could provide insights into the azole resistance mechanisms operating in fungi. The primers used here may be useful molecular tools for facilitating the cloning of novel 14-α sterol demethylase genes in other filamentous fungi.
doi:10.1128/JCM.39.7.2431-2438.2001
PMCID: PMC88166  PMID: 11427550
12.  Genetic Basis for Differential Activities of Fluconazole and Voriconazole against Candida krusei 
Invasive infections caused by Candida krusei are a significant concern because this organism is intrinsically resistant to fluconazole. Voriconazole is more active than fluconazole against C. krusei in vitro. One mechanism of fluconazole resistance in C. krusei is diminished sensitivity of the target enzyme, cytochrome P450 sterol 14α-demethylase (CYP51), to inhibition by this drug. We investigated the interactions of fluconazole and voriconazole with the CYP51s of C. krusei (ckCYP51) and fluconazole-susceptible Candida albicans (caCYP51). We found that voriconazole was a more potent inhibitor of both ckCYP51 and caCYP51 in cell extracts than was fluconazole. Also, the ckCYP51 was less sensitive to inhibition by both drugs than was caCYP51. These results were confirmed by expressing the CYP51 genes from C. krusei and C. albicans in Saccharomyces cerevisiae and determining the susceptibility of the transformants to voriconazole and fluconazole. We constructed homology models of the CYP51s of C. albicans and C. krusei based on the crystal structure of CYP51 from Mycobacterium tuberculosis. These models predicted that voriconazole is a more potent inhibitor of both caCYP51 and ckCYP51 than is fluconazole, because the extra methyl group of voriconazole results in a stronger hydrophobic interaction with the aromatic amino acids in the substrate binding site and more extensive filling of this site. Although there are multiple differences in the predicted amino acid sequence of caCYP51 and ckCYP51, the models of the two enzymes were quite similar and the mechanism for the relative resistance of ckCYP51 to the azoles was not apparent.
doi:10.1128/AAC.47.4.1213-1219.2003
PMCID: PMC152512  PMID: 12654649
13.  Utility of a Phylogenetic Perspective in Structural Analysis of CYP72A Enzymes from Flowering Plants 
PLoS ONE  2016;11(9):e0163024.
Plant adaptation to external pressures depends on functional diversity in cytochrome P450 (CYP) enzymes. CYPs contain structural domains necessary for the characteristic P450 fold that allows monooxygenation, but they also have great variation in substrate binding affinity. Plant genomes typically contain hundreds of CYPs that contribute to essential functions and species-specific metabolism. The CYP72A subfamily is conserved in angiosperms but its contribution to physiological functions is largely unknown. With genomic information available for many plants, a focused analysis of CYP subfamily diversity is important to understand the contributions of these enzymes to plant evolution. This study examines the extent to which independent gene duplication and evolution have contributed to structural diversification of CYP72A enzymes in different plant lineages. CYP72A genes are prevalent across angiosperms, but the number of genes within each genome varies greatly. The prevalence of CYP72As suggest that the last common ancestor of flowering plants contained a CYP72A sequence, but gene duplication and retention has varied greatly for this CYP subfamily. Sequence comparisons show that CYP72As are involved in species-specific metabolic functions in some plants while there is likely functional conservation between closely related species. Analysis of structural and functional domains within groups of CYP72As reveals clade-specific residues that contribute to functional constraints within subsets of CYP72As. This study provides a phylogenetic framework that allows comparisons of structural features within subsets of the CYP72A subfamily. We examined a large number of sequences from a broad collection of plant species to detect patterns of functional conservation across the subfamily. The evolutionary relationships between CYPs in plant genomes are an important component in understanding the evolution of biochemical diversity in plants.
doi:10.1371/journal.pone.0163024
PMCID: PMC5036807  PMID: 27669508
14.  Structural characterization of CYP144A1 – a cytochrome P450 enzyme expressed from alternative transcripts in Mycobacterium tuberculosis 
Scientific Reports  2016;6:26628.
Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis (TB). The virulent Mtb H37Rv strain encodes 20 cytochrome P450 (CYP) enzymes, many of which are implicated in Mtb survival and pathogenicity in the human host. Bioinformatics analysis revealed that CYP144A1 is retained exclusively within the Mycobacterium genus, particularly in species causing human and animal disease. Transcriptomic annotation revealed two possible CYP144A1 start codons, leading to expression of (i) a “full-length” 434 amino acid version (CYP144A1-FLV) and (ii) a “truncated” 404 amino acid version (CYP144A1-TRV). Computational analysis predicted that the extended N-terminal region of CYP144A1-FLV is largely unstructured. CYP144A1 FLV and TRV forms were purified in heme-bound states. Mass spectrometry confirmed production of intact, His6-tagged forms of CYP144A1-FLV and -TRV, with EPR demonstrating cysteine thiolate coordination of heme iron in both cases. Hydrodynamic analysis indicated that both CYP144A1 forms are monomeric. CYP144A1-TRV was crystallized and the first structure of a CYP144 family P450 protein determined. CYP144A1-TRV has an open structure primed for substrate binding, with a large active site cavity. Our data provide the first evidence that Mtb produces two different forms of CYP144A1 from alternative transcripts, with CYP144A1-TRV generated from a leaderless transcript lacking a 5′-untranslated region and Shine-Dalgarno ribosome binding site.
doi:10.1038/srep26628
PMCID: PMC4880925  PMID: 27225995
15.  Trypanosoma cruzi CYP51 Inhibitor Derived from a Mycobacterium tuberculosis Screen Hit 
Background
The two front-line drugs for chronic Trypanosoma cruzi infections are limited by adverse side-effects and declining efficacy. One potential new target for Chagas' disease chemotherapy is sterol 14α-demethylase (CYP51), a cytochrome P450 enzyme involved in biosynthesis of membrane sterols.
Methodology/Principal Finding
In a screening effort targeting Mycobacterium tuberculosis CYP51 (CYP51Mt), we previously identified the N-[4-pyridyl]-formamide moiety as a building block capable of delivering a variety of chemotypes into the CYP51 active site. In that work, the binding modes of several second generation compounds carrying this scaffold were determined by high-resolution co-crystal structures with CYP51Mt. Subsequent assays against the CYP51 orthologue in T. cruzi, CYP51Tc, demonstrated that two of the compounds tested in the earlier effort bound tightly to this enzyme. Both were tested in vitro for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. Cure of treated host cells was confirmed by prolonged incubation in the absence of the inhibiting compound. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability (phenylalanine versus isoleucine) of a single residue at a critical position in the active site.
Conclusions/Significance
CYP51Mt-based crystal structure analysis revealed that the functional groups of the two tightly bound compounds are likely to occupy different spaces in the CYP51 active site, suggesting the possibility of combining the beneficial features of both inhibitors in a third generation of compounds to achieve more potent and selective inhibition of CYP51Tc.
Author Summary
Enzyme sterol 14α-demethylase (CYP51) is a well-established target for anti-fungal therapy and is a prospective target for Chagas' disease therapy. We previously identified a chemical scaffold capable of delivering a variety of chemical structures into the CYP51 active site. In this work the binding modes of several second generation compounds carrying this scaffold were determined in high-resolution co-crystal structures with CYP51 of Mycobacterium tuberculosis. Subsequent assays against CYP51 in Trypanosoma cruzi, the agent of Chagas' disease, demonstrated that two of the compounds bound tightly to the enzyme. Both were tested for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. This compound is currently being evaluated in animal models of Chagas' disease. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability of a single amino acid residue at a critical position in the active site. Our work is aimed at rational design of potent and highly selective CYP51 inhibitors with potential to become therapeutic drugs. Drug selectivity to prevent host–pathogen cross-reactivity is pharmacologically important, because CYP51 is present in human host.
doi:10.1371/journal.pntd.0000372
PMCID: PMC2629123  PMID: 19190730
16.  Computational identification and binding analysis of orphan human cytochrome P450 4X1 enzyme with substrates 
BMC Research Notes  2015;8:9.
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/s13104-015-0976-4
PMCID: PMC4322450  PMID: 25595103
Homology modeling; Human cytochrome; CYP4X1; Molecular docking; Arachidonic acid; Anandamide
17.  Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3 
Cytochrome P450 monooxygenases (P450s), which constitute a superfamily of heme-containing proteins, catalyze the direct oxidation of a variety of compounds in a regio- and stereospecific manner; therefore, they are promising catalysts for use in the oxyfunctionalization of chemicals. In the course of our comprehensive substrate screening for all 27 putative P450s encoded by the Streptomyces griseus genome, we found that Escherichia coli cells producing an S. griseus P450 (CYP154C3), which was fused C terminally with the P450 reductase domain (RED) of a self-sufficient P450 from Rhodococcus sp., could transform various steroids (testosterone, progesterone, Δ4-androstene-3,17-dione, adrenosterone, 1,4-androstadiene-3,17-dione, dehydroepiandrosterone, 4-pregnane-3,11,20-trione, and deoxycorticosterone) into their 16α-hydroxy derivatives as determined by nuclear magnetic resonance and high-resolution mass spectrometry analyses. The purified CYP154C3, which was not fused with RED, also catalyzed the regio- and stereospecific hydroxylation of these steroids at the same position with the aid of ferredoxin and ferredoxin reductase from spinach. The apparent equilibrium dissociation constant (Kd) values of the binding between CYP154C3 and these steroids were less than 8 μM as determined by the heme spectral change, indicating that CYP154C3 strongly binds to these steroids. Furthermore, kinetic parameters of the CYP154C3-catalyzed hydroxylation of Δ4-androstene-3,17-dione were determined (Km, 31.9 ± 9.1 μM; kcat, 181 ± 4.5 s−1). We concluded that CYP154C3 is a steroid D-ring 16α-specific hydroxylase which has considerable potential for industrial applications. This is the first detailed enzymatic characterization of a P450 enzyme that has a steroid D-ring 16α-specific hydroxylation activity.
doi:10.1128/AEM.03504-13
PMCID: PMC3911049  PMID: 24334658
18.  Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9 
PLoS Computational Biology  2011;7(8):e1002152.
The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme's buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.
Author Summary
We describe the first atomic-detail models and simulations of a full-length, membrane-bound mammalian cytochrome P450. To date, all the structural studies of microsomal, drug-metabolizing cytochrome P450s have been performed using engineered, solubilized forms of the enzymes and it is not yet understood how the membrane influences their structure, dynamics, and ability to bind substrates. We focused on CYP2C9, the second most abundant cytochrome P450 in the human liver which oxidizes 20% of all marketed drugs. Here, we have derived models of CYP2C9-membrane complexes with a modeling procedure based on molecular dynamics simulations started with arbitrary configurations of the protein in the membrane and performed using both coarse grained and atomic-detail molecular representations. This procedure is expected to be generally applicable to proteins that are anchored in the membrane with a single transmembrane helix. The models and simulations provide evidence for selective substrate access and product release routes in membrane-bound CYPs. This observation may contribute to new strategies to manipulate the activity of CYPs and other enzymes with buried active sites. We anticipate that this study will bring about a paradigm shift towards studying microsomal CYPs as dynamic structures in their natural, lipid environment rather than in artificially solubilized forms.
doi:10.1371/journal.pcbi.1002152
PMCID: PMC3154944  PMID: 21852944
19.  The Cytochrome P450 Superfamily Complement (CYPome) in the Annelid Capitella teleta 
PLoS ONE  2014;9(11):e107728.
The Cytochrome P450 super family (CYP) is responsible for a wide range of functions in metazoans, having roles in both exogenous and endogenous substrate metabolism. Annelids are known to metabolize polycyclic aromatic hydrocarbons (PAHs) and produce estrogen. CYPs are postulated to be key enzymes in these processes in annelids. In this study, the CYP complement (CYPome) of the annelid Capitella teleta has been robustly identified and annotated with the genome assembly available. Phylogenetic analyses were performed to understand the evolutionary relationships between CYPs in C. teleta and other species. Predictions of which CYPs are potentially involved in both PAH metabolism and steroidogensis were made based on phylogeny. Annotation of 84 full length and 12 partial CYP sequences predicted a total of 96 functional CYPs in C. teleta. A further 13 CYP fragments were found but these may be pseudogenes. The C. teleta CYPome contained 24 novel CYP families and seven novel CYP subfamilies within existing families. A phylogenetic analysis identified that the C. teleta sequences were found in 9 of the 11 metazoan CYP clans. Two CYPs, CYP3071A1 and CYP3072A1, did not cluster with any metazoan CYP clans. We found xenobiotic response elements (XREs) upstream of C. teleta CYPs related to vertebrate CYP1 (CYP3060A1, CYP3061A1) and from families with reported transcriptional upregulation in response to PAH exposure (CYP4, CYP331). C. teleta had a CYP51A1 with ∼65% identity to vertebrate CYP51A1 sequences and has been predicted to have lanosterol 14 α-demethylase activity. CYP376A1, CYP3068A1, CYP3069A1, and CYP3070A1 were the most appropriate candidates for steroidogenesis genes based on their phylogeny and warrant further analyses, though no specific aromatase (estrogen synthesis) candidates were found. Presence of XREs upstream of C. teleta CYPs may indicate a functional aryl hydrocarbon receptor in C. teleta and candidate CYPs for studies of PAH metabolism.
doi:10.1371/journal.pone.0107728
PMCID: PMC4229089  PMID: 25390889
20.  Identification and functional characterization of the CYP51 gene from the yeast Xanthophyllomyces dendrorhous that is involved in ergosterol biosynthesis 
BMC Microbiology  2015;15:89.
Background
Xanthophyllomyces dendrorhous is a basidiomycetous yeast that synthesizes astaxanthin, a carotenoid with great biotechnological impact. The ergosterol and carotenoid synthetic pathways derive from the mevalonate pathway and involve cytochrome P450 enzymes. Among these enzymes, the CYP51 family, which is involved in ergosterol biosynthesis, is one of the most remarkable that has C14-demethylase activity.
Results
In this study, the CYP51 gene from X. dendrorhous was isolated and its function was analyzed. The gene is composed of ten exons and encodes a predicted 550 amino acid polypeptide that exhibits conserved cytochrome P450 structural characteristics and shares significant identity with the sterol C14-demethylase from other fungi. The functionality of this gene was confirmed by heterologous complementation in S. cerevisiae. Furthermore, a CYP51 gene mutation in X. dendrorhous reduced sterol production by approximately 40% and enhanced total carotenoid production by approximately 90% compared to the wild-type strain after 48 and 120 h of culture, respectively. Additionally, the CYP51 gene mutation in X. dendrorhous increased HMGR (hydroxy-methylglutaryl-CoA reductase, involved in the mevalonate pathway) and crtR (cytochrome P450 reductase) transcript levels, which could be associated with reduced ergosterol production.
Conclusions
These results suggest that the CYP51 gene identified in X. dendrorhous encodes a functional sterol C14-demethylase that is involved in ergosterol biosynthesis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0428-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12866-015-0428-2
PMCID: PMC4415319  PMID: 25906980
Cytochrome P450 enzyme systems (P450); Astaxanthin; Sterols; Sterol C14-demethylase
21.  Molecular cloning and expression patterns of the cholesterol side chain cleavage enzyme (CYP11A1) gene during the reproductive cycle in goose (Anas cygnoides) 
Background
CYP11A1, a gene belonging to the family 11 of cytochrome P450, encodes a crucial steroidogenic enzyme that catalyzes the initial step in the production of all classes of steroids. Many studies show that CYP11A1 plays a role in ovary function. However, the role of CYP11A1 in goose reproductive cycle remains largely unknown.
Results
In this study, full-length CYP11A1 cDNA of Zhedong goose was obtained using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA consisted of a 96-base pair (bp) 5′untranslated region (UTR), a 179-bp 3′UTR and a 1509-bp open reading frame. The open reading frame encodes a putative 503 amino acid protein that shares high homology with CYP11A1 of other birds. The amino acid sequence possesses conserved domains of the P450 superfamily, which include the steroid-binding domain and the heme-binding region. Real-time quantitative polymerase chain reaction (qPCR) analysis revealed CYP11A1 mRNA was expressed ubiquitously in every Zhedong goose tissue analyzed, including the heart, liver, glandular stomach, lung, spleen, kidney, intestinum tenue, intestinum crassum, cerebrum, cerebellum, muscle, oviduct, pituitary, hypothalamus and ovary.. The relatively low levels of CYP11A1 mRNA were detected in pituitary, ovary and oviduct tissues at ovulation when compared with levels at oviposition. Interestingly, higher expression was observed in ovary and oviduct tissues during brooding. Lastly, higher mRNA expression of Yangzhou geese was detected during the ovulation period than that of Zhedong geese.
Conclusions
Our findings reveal the sequence characterization and expression patterns of the CYP11A1 gene during the goose reproductive cycle, which may provides correlative evidence that CYP11A1 expression is important in reproduction activity.
doi:10.1186/s40104-015-0053-9
PMCID: PMC4688999  PMID: 26702355
CYP11A1; Gene expression; Goose; Reproduction
22.  Hydrogen-Deuterium Exchange Mass Spectrometry for Investigation of Backbone Dynamics of Oxidized and Reduced Cytochrome P450cam 
Journal of inorganic biochemistry  2007;102(2):364-370.
Backbone dynamics of the camphor monoxygenase cytochrome P450cam (CYP101) as a function of oxidation/ligation state of the heme iron were investigated via hydrogen/deuterium exchange (H/D exchange) as monitored by mass spectrometry. Main chain amide NH hydrogens can exchange readily with solvent and the rate of this exchange depends upon, among other things, dynamic fluctuations in local structural elements. A fluxional region of the polypeptide will exchange more quickly with solvent than one that is more constrained. In most regions of the enzyme, exchange rates were similar between oxidized high-spin camphor-bound and reduced camphor-and CO-bound CYP101 (CYP-S and CYP-S-CO, respectively.) However, in regions of the protein that have previously been implicated in substrate access by structural and molecular dynamics investigations, the reduced enzyme shows significantly slower exchange rates than the oxidized CYP-S. This observation corresponds to increased flexibility of the oxidized enzyme relative to the reduced form. Structural features previously found to be perturbed in CYP-S-CO upon binding of the biologically relevant effector and reductant putidaredoxin (Pdx) as determined by nuclear magnetic resonance are also more protected from exchange in the reduced state. To our knowledge, this study represents the first experimental investigation of backbone dynamics within the P450 family using this methodology.
doi:10.1016/j.jinorgbio.2007.10.001
PMCID: PMC2279175  PMID: 18023482
redox-dependent dynamics; electron transfer; tandem mass spectrometry; hydrogen/deuterium exchange
23.  The Schistosoma mansoni Cytochrome P450 (CYP3050A1) Is Essential for Worm Survival and Egg Development 
PLoS Neglected Tropical Diseases  2015;9(12):e0004279.
Schistosomiasis affects millions of people in developing countries and is responsible for more than 200,000 deaths annually. Because of toxicity and limited spectrum of activity of alternatives, there is effectively only one drug, praziquantel, available for its treatment. Recent data suggest that drug resistance could soon be a problem. There is therefore the need to identify new drug targets and develop drugs for the treatment of schistosomiasis. Analysis of the Schistosoma mansoni genome sequence for proteins involved in detoxification processes found that it encodes a single cytochrome P450 (CYP450) gene. Here we report that the 1452 bp open reading frame has a characteristic heme-binding region in its catalytic domain with a conserved heme ligating cysteine, a hydrophobic leader sequence present as the membrane interacting region, and overall structural conservation. The highest sequence identity to human CYP450s is 22%. Double stranded RNA (dsRNA) silencing of S. mansoni (Sm)CYP450 in schistosomula results in worm death. Treating larval or adult worms with antifungal azole CYP450 inhibitors results in worm death at low micromolar concentrations. In addition, combinations of SmCYP450-specific dsRNA and miconazole show additive schistosomicidal effects supporting the hypothesis that SmCYP450 is the target of miconazole. Treatment of developing S. mansoni eggs with miconazole results in a dose dependent arrest in embryonic development. Our results indicate that SmCYP450 is essential for worm survival and egg development and validates it as a novel drug target. Preliminary structure-activity relationship suggests that the 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol moiety of miconazole is necessary for activity and that miconazole activity and selectivity could be improved by rational drug design.
Author Summary
Over 600 million people in endemic countries are at risk of contracting schistosomiasis, which results in over 200,000 deaths each year and significant illness to most people that are infected. There are concerns that the drug widely used for the treatment of schistosomiasis, praziquantel, may be losing efficacy due to evolution of drug resistant worms. Since the disease mainly affects the poor in developing countries, pharmaceutical companies have little interest in developing new drugs and none are currently being tested. In this paper we focus on a novel parasite protein, cytochrome P450, which we propose to be a new drug target. Worms are unusual in having only one cytochrome P450 gene; humans have 57 cytochrome P450 genes. By using reverse genetic and chemical approaches we found that the schistosome cytochrome P450 is essential for worm survival and egg development and, therefore, is an essential and druggable target. Drugs that target fungal cytochrome P450s and are already in use for treating several human diseases were identified as potential hits for further development for schistosomiasis treatment.
doi:10.1371/journal.pntd.0004279
PMCID: PMC4694641  PMID: 26713732
24.  Rapid Birth–Death Evolution Specific to Xenobiotic Cytochrome P450 Genes in Vertebrates 
PLoS Genetics  2007;3(5):e67.
Genes vary greatly in their long-term phylogenetic stability and there exists no general explanation for these differences. The cytochrome P450 (CYP450) gene superfamily is well suited to investigating this problem because it is large and well studied, and it includes both stable and unstable genes. CYP450 genes encode oxidase enzymes that function in metabolism of endogenous small molecules and in detoxification of xenobiotic compounds. Both types of enzymes have been intensively studied. My analysis of ten nearly complete vertebrate genomes indicates that each genome contains 50–80 CYP450 genes, which are about evenly divided between phylogenetically stable and unstable genes. The stable genes are characterized by few or no gene duplications or losses in species ranging from bony fish to mammals, whereas unstable genes are characterized by frequent gene duplications and losses (birth–death evolution) even among closely related species. All of the CYP450 genes that encode enzymes with known endogenous substrates are phylogenetically stable. In contrast, most of the unstable genes encode enzymes that function as xenobiotic detoxifiers. Nearly all unstable CYP450 genes in the mouse and human genomes reside in a few dense gene clusters, forming unstable gene islands that arose by recurrent local gene duplication. Evidence for positive selection in amino acid sequence is restricted to these unstable CYP450 genes, and sites of selection are associated with substrate-binding regions in the protein structure. These results can be explained by a general model in which phylogenetically stable genes have core functions in development and physiology, whereas unstable genes have accessory functions associated with unstable environmental interactions such as toxin and pathogen exposure. Unstable gene islands in vertebrates share some functional properties with bacterial genomic islands, though they arise by local gene duplication rather than horizontal gene transfer.
Author Summary
Genes vary greatly in their long-term phylogenetic stability, and there exists no general explanation for these differences. Stable genes persist as a single copy over a wide range of distantly related species, whereas unstable genes undergo frequent duplication and loss in a process called birth-death evolution. The vertebrate cytochrome P450 (CYP450) gene superfamily includes many genes that are present in a single copy in species ranging from teleost fish to mammals and other groups of genes that undergo active birth-death evolution across the same species. The author found that nearly all stable CYP450 genes encode enzymes known to function in the synthesis and degradation of steroid and retinoid hormones (and related molecules). These hormones function in core developmental pathways in vertebrates. In contrast, most unstable CYP450 genes encode enzymes that detoxify foreign small molecules (called xenobiotics—foreign biochemicals). In addition, many of the unstable CYP450 genes are subject to natural selection to change their amino acid sequence over time (positive selection), probably in response to changes in xenobiotic exposure. These findings suggest that stable and unstable genes differ in their rates of birth-death evolution, because stable genes have core endogenous functions whereas unstable genes respond to changing environmental conditions.
doi:10.1371/journal.pgen.0030067
PMCID: PMC1866355  PMID: 17500592
25.  Insights into drug metabolism by cytochromes P450 from modelling studies of CYP2D6-drug interactions 
British Journal of Pharmacology  2007;153(Suppl 1):S82-S89.
The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolizing CYPs provide crucial protection from the effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Ideally, the information on the possible metabolism by CYPs required during drug development would be obtained from crystal structures of all the CYPs of interest. For some years only crystal structures of distantly related bacterial CYPs were available and homology modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain useful functional information. A significant step forward in the reliability of these models came seven years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of six human enzymes, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. In this review we describe as a case study the evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism. This work has led directly to the successful design of CYP2D6 mutants with novel activity—including creating a testosterone hydroxylase, converting quinidine from inhibitor to substrate, creating a diclofenac hydroxylase and creating a dextromethorphan O-demethylase. Our modelling-derived hypothesis-driven integrated interdisciplinary studies have given key insight into the molecular determinants of CYP2D6 and other important drug metabolizing enzymes.
doi:10.1038/sj.bjp.0707570
PMCID: PMC2268062  PMID: 18026129
comparative modelling; cytochromes P450; CYP2D6; docking; drug metabolism; homology modelling; hypothesis-driven studies

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