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.
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
The cytochrome P450 sterol 14α-demethylase (CYP51) of Candida albicans is involved in an essential step of ergosterol biosynthesis and is the target for azole antifungal compounds. We have undertaken site-directed mutation of C. albicans CYP51 to produce a recombinant mutant protein with the amino acid substitution R467K corresponding to a mutation observed clinically. This alteration perturbed the heme environment causing an altered reduced-carbon monoxide difference spectrum with a maximum at 452 nm and reduced the affinity of the enzyme for fluconazole, as shown by ligand binding studies. The specific activity of CYP51(R467K) for the release of formic acid from 3β-[32-3H]hydroxylanost-7-en-32-ol was 70 pmol/nmol of P450/min for microsomal protein compared to 240 pmol/nmol of P450/min for microsomal fractions expressing wild-type CYP51. Furthermore, inhibition of activity by fluconazole revealed a 7.5-fold-greater azole resistance of the recombinant protein than that of the wild type. This study demonstrates that resistance observed clinically can result from the altered azole affinity of the fungal CYP51 enzyme.
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.
Cytochromes P450 (CYPs) are a super family of heme-containing enzymes well-known for their monooxgenase reaction. There are
57 CYP isoenzymes found in human which exhibit specific physiological functions. Thirteen members of this super family are
classified as “orphan” CYP because of their unknown enzymatic functions. CYP4V2 is found to be a potential drug target for Bietti
crystalline corneoretinal dystrophy (BCD). However, three-dimensional structure, the active site topology and substrate binding
modes of CYP4V2 remain unclear. In this study, the three-dimensional model of CYP4V2 was constructed using the homology
modeling method. Four possible fatty acid substrates namely, caprylic, lauric, myrisitc and palmitic acids were optimized and
evaluated for drug likeness using Lipinski's rule of five. Further, these substrates were docked into active sites of CYP4V2 and
several key residues responsible for substrate binding were identified. These findings will be helpful for the structure-based drug
design and detailed characterization of the biological roles of CYP4V2.
Human Cytochrome; P450 4V2; CYP4V2; homology modeling; molecular docking; fatty acid substrates; ligand binding site; caprylic acid; lauric acid; myristic acid and palmitic acid
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.
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.
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.
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.
The cytochrome P450 sterol 14α-demethylase enzyme (CYP51) is the target of azole antifungals. Azoles block ergosterol synthesis, and thereby fungal growth, by binding in the active-site cavity of the enzyme and ligating the iron atom of the heme cofactor through a nitrogen atom of the azole. Mutations in and around the CYP51 active site have resulted in azole resistance. In this work, homology models of the CYP51 enzymes from Aspergillus fumigatus and Candida albicans were constructed based on the X-ray crystal structure of CYP51 from Mycobacterium tuberculosis. Using these models, binding modes for voriconazole (VOR), fluconazole (FLZ), itraconazole (ITZ), and posaconazole (POS) were predicted from docking calculations. Previous work had demonstrated that mutations in the vicinity of the heme cofactor had a greater impact on the binding of FLZ and VOR than on the binding of POS and ITZ. Our modeling data suggest that the long side chains of POS and ITZ occupy a specific channel within CYP51 and that this additional interaction, which is not available to VOR and FLZ, serves to stabilize the binding of these azoles to the mutated CYP51 proteins. The model also predicts that mutations that were previously shown to specifically impact POS susceptibility in A. fumigatus and C. albicans act by interfering with the binding of the long side chain.
Cytochrome P450s (CYP450s) are hemoproteins catalysing diverse biochemical reactions important for metabolism of xenobiotics and synthesis of physiologically important compounds such as sterols. Therefore, they are functionally important for survival of invading pathogens. One such opportunistic pathogen Leishmania donovani causes visceral leishmaniasis worldwide, which is an important public health problem due to significant disease burden. The parasite genome database, Gene DB, annotates 3 CYP450s in Leishmania, however, the functional role of cytochrome P450 enzymes in Leishmania spp. remains elusive.
A CYP450-like gene cloned from Leishmania donovani was identified as a novel CYP450, the CYP5122A1. Upon co-localization with organelle specific markers, CYP5122A1 distribution was shown to be localized in the promastigote ER, mitochondria and the glycosomes. Replacement of one allele of CYP5122A1 with either neomycin or hygromycin gene by homologous recombination in Leishmania promastigotes induced substantial reduction of CYP5122A1 expression. These parasites showed impaired growth, lower mitochondrial Ca2+ and membrane potential resulting in low ATP generation. Also, these parasites were less infective in vitro and in vivo than their wild-type counterparts as assessed by incubation of Leishmania promastigotes with macrophages in vitro as well as through administration of parasites into hamsters. The HKOs were more susceptible to drugs like miltefosine and antimony, but showed reduced sensitivity to amphotericin B. Removal of two alleles of CYP5122A1 did not allow the parasites to survive. The mutant parasites showed 3.5 times lower ergosterol level as compared to the wild-type parasites when estimated by Gas chromatography/mass spectrometry. Complementation of CYP5122A1 through episomal expression of protein by using pXG-GFP+2 vector partially rescued CYP5122A1 expression and restored ergosterol levels by 1.8 times. Phenotype reversal included restored growth pattern and lesser drug susceptibility.
In summary, this study establishes CYP5122A1 as an important molecule linked to processes like cell growth, infection and ergosterol biosynthesis in Leishmania donovani.
Structural perturbations in cytochrome P450cam (CYP101) induced by the soluble fragment of rate cytochrome b5, a non-physiological effector of CYP101, were investigated by NMR spectroscopy and compared with the perturbations induced by the physiological reductant and effector, putidaredoxin (Pdx). Chemical shifts of perdeuterated [U-15N] CYP101 backbone amide (NH) resonances were monitored as a function of cytochrome b5 concentration by 1H, 15N TROSY-HSQC experiments. The association of cytochrome b5 with the reduced CYP101-camphor-carbon monoxide complex (CYP-S-CO) perturbs many of the same resonances that Pdx does, including regions of the CYP101 molecule implicated in substrate access and orientation. The perturbations are smaller in magnitude than those observed with Pdxr due to a lower binding affinity (Kd = 13 ± 3 mM, for reduced cytochrome b5-CYP-S-CO complex compared to Kd = 26 ± 12 μM for the Pdx-CYP-S-CO complex). The results are in accord with our previous suggestion that the observed perturbations are related to effector activity and support the proposal that the primary role of the effector is to populate the active conformation of CYP101 to prevent uncoupling [Pochapsky et al. Biochemistry 42, 5649-5656 (2003)]. A titratable perturbation is observed at the 1H resonance of the 8-CH3 of CYP101-bound camphor upon addition of cytochrome b5, a phenomenon also associated with the formation of CYP101·Pdx complex albeit with larger perturbations [Wei et al., J. Am. Chem. Soc. 127, 6974-6 976 (2005)]. The effector activity of the particular rat cytochrome b5 construct used for NMR studies was confirmed by monitoring the enzymatic turnover to yield 5-exo-hydroxy camphor using gas chromatography/mass spectrometry. Finally, the common features of the perturbations observed in the NMR spectra of the two complexes are discussed and their relevance to effector activity considered.
cytochrome P450; cytochrome b5; putidaredoxin; NMR; effector
Cytochrome P450s are superfamily of heme proteins which generally monooxygenate hydrophobic compounds. The human
cytochrome P450 4F22 (CYP4F22) was categorized into “orphan” CYPs because of its unknown function. CYP4F22 is a potential
drug target for cancer therapy. However, three-dimensional structure, the active site topology and substrate specificity of CYP4F22
remain unclear. In this study, a three-dimensional model of human P450 4F22 was constructed by comparative modeling using
Modeller 9v5. The resulting model was refined by energy minimization subjected to the quality assessment from both geometric
and energetic aspects and was found to be of reasonable quality. Docking approach was employed to dock arachidonic acid into
the active site of CYP4F22 in order to probe the ligand-binding modes. As a result, several key residues were identified to be
responsible for the binding of arachidonic acid with CYP4F22. These findings provide useful information for understanding the
biological roles of CYP4F22 and structure-based drug design.
Human Cytochrome; P450 4F22; CYP4F22; comparative modeling; molecular docking; arachidonic acid; ligand binding site; 2D QSAR
Cytochrome P450 (CYP) 3A4 contributes to the metabolism of approximately 50% of commercial drugs by oxidizing a large number of structurally diverse substrates. Like other endoplasmic reticulum-localized P450s, CYP3A4 contains a membrane-anchoring N-terminal helix and a significant number of hydrophobic domains, important for the interaction between CYP3A4 and the membrane. Although the membrane affects specificity of CYP3A4 ligand binding, the structural details of the interaction have not been revealed so far because x-ray crystallography studies are available only for the soluble domain of CYP3A4. Here we report sample preparation and initial magic-angle spinning (MAS) solid-state NMR (SSNMR) of CYP3A4 (Δ3−12) embedded in a nanoscale membrane bilayer, or Nanodisc. The growth protocol yields ∼2.5 mg of the enzymatically active, uniformly 13C, 15N-enriched CYP3A4 from a liter of growth medium. Polyethylene glycol 3350-precipitated CYP3A4 in Nanodiscs yields spectra of high resolution and sensitivity, consistent with a folded, homogeneous protein. CYP3A4 in Nanodiscs remains enzymatically active throughout the precipitation protocol as monitored by bromocriptine binding. The 13C line widths measured from 13C-13C 2D chemical shift correlation spectra are ∼0.5 ppm. The secondary structure distribution within several amino acid types determined from 13C chemical shifts is consistent with the ligand-free x-ray structures. These results demonstrate that MAS SSNMR can be performed on Nanodisc-embedded membrane proteins in a folded, active state. The combination of SSNMR and Nanodisc methodologies opens up new possibilities for obtaining structural information on CYP3A4 and other integral membrane proteins with full retention of functionality.
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.
Inhibition of sterol-14α-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51, although selective inhibitors of the human target are also of interest as anticholesterol agents. A strain of Saccharomyces cerevisiae that was humanized with respect to the amino acids encoded at the CYP51 (ERG11) yeast locus (BY4741:huCYP51) was produced. The strain was validated with respect to gene expression, protein localization, growth characteristics, and sterol content. The MIC was determined and compared to that for the wild-type parental strain (BY4741), using clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole. The humanized strain showed up to >1,000-fold-reduced susceptibility to the orally active azole drugs, while the topical agents showed no difference. Data from growth kinetic measurements substantiated this finding but also revealed reduced effectiveness against the humanized strain for the topical drugs. Cellular sterol profiles reflected the decreased susceptibility of BY4741:huCYP51 and showed a smaller depletion of ergosterol and accumulation of 14α-methyl-ergosta-8, 24(28)-dien-3β-6α-diol than the parental strain under the same treatment conditions. This strain provides a useful tool for initial specificity testing for new drugs targeting CYP51 and clearly differentiates azole antifungals in a side-by-side comparison.
Cytochrome P450 46A1 (CYP46A1) and NADPH-cytochrome P450 oxidoreductase (CPR) are the components of the brain microsomal mixed-function monooxygenase system that catalyzes the conversion of cholesterol to 24-hydroxycholesterol. Both CYP46A1 and CPR are monotopic membrane proteins that are anchored to the endoplasmic reticulum via the N-terminal transmembrane domain. The exact mode of peripheral association of CYP46A1 and CPR with the membrane is unknown. Therefore, we studied their membrane topology by using an approach in which solution-exposed portion of heterologously expressed membrane-bound CYP46A1 or CPR was removed by digestion with either trypsin or chymotrypsin followed by extraction of the residual peptides and their identification by mass spectrometry. The identified putative membrane-interacting peptides were mapped onto available crystal structures of CYP46A1 and CPR and the proteins were positioned in the membrane considering spatial location of the missed cleavage sites located within these peptide as well as the flanking residues whose cleavage produced these peptides. Experiments were then carried out to validate the inference from our studies that the substrate, cholesterol, enters CYP46A1 from the membrane. As for CPR, its putative membrane topology indicates that the Q153R and R316W missense mutations found in patients with disordered steroidogenesis are located within the membrane-associated regions. This information may provide insight in the deleterious nature of these mutations.
CYP46A1; CPR; Crystal structure; MALDI; Membrane topology
Tipifarnib (R115777), an inhibitor of human protein farnesyltransferase (PFT), is shown to be a highly potent inhibitor of Trypanosoma cruzi growth (ED50 = 4 nM). Surprisingly, this is due to the inhibition of cytochrome P450 sterol 14-demethylase (CYP51, EC 18.104.22.168). Homology models of the T. cruzi CYP51 were used for the prediction of the binding modes of the substrate lanosterol and of Tipifarnib, providing a basis for the design of derivatives with selectivity for TcCYP51 over human PFT.
Estrogen synthesis evolved in chordates to control reproduction. The terminal enzyme in the cascade directly responsible for estrogen synthesis is aromatase cytochrome P450 (P450arom) encoded by the CYP19 gene. Mammals typically have a single CYP19 gene but pigs, peccaries and other Suiformes have two or more resulting from duplication in a common ancestor. Duplication of CYP genes in the steroid synthetic cascade has occurred for only one other enzyme, also terminal, 11β-hydroxylase P450 (P450c11). P450arom and P450c11 share common substrates and even physiological functions as possible remnants from a common P450 progenitor, perhaps an ancestral P450arom, which is supported by phylogenetic analysis. Conserved tissue-specific expression patterns of P450arom paralogs in placenta and gonads of pigs and peccaries suggest how functional adaptation may have proceeded divergently and influenced adopted reproductive strategies including ovulation rate and litter size. Data suggest that the porcine placental paralog evolved catalytically to protect female conceptuses from testosterone produced by male siblings; the gonadal paralog to synthesize a novel, nonaromatizable testosterone metabolite (1OH-testosterone) that may increase ovulation rate. This would represent a coevolution facilitating litter bearing as pigs diverged from peccaries. Evidence of convergence between the peccary CYP19 genes and lower tissue expression may therefore represent initiation of loss of the functional paralogs. Studies on the Suiforme aromatases provide insights into the evolution of the steroidogenic cascade and metabolic pathways in general, how it translates into physiological adaptations (altered reproductive strategies for instance), and how duplicated genes become stabilized or disappear from genomes.
Sterol 14α-demethylases (CYP51) serve as primary targets for antifungal drugs and specific inhibition of CYP51s in protozoan parasites Trypanosoma brucei (TB) and Trypanosoma cruzi (TC) might provide an effective treatment strategy for human trypanosomiases. Primary inhibitor selection is based initially on the cytochrome P450 spectral response to ligand binding. Ligands which demonstrate strongest binding parameters were examined as inhibitors of reconstituted TB and TC CYP51 activity in vitro. Direct correlation between potency of the compounds as CYP51 inhibitors and their antiparasitic effect in TB and TC cells implies essential requirements for endogenous sterol production in both trypanosomes and suggests a novel lead structure with a defined region most promising for further modifications. The approach developed here can be used for further large-scale search for new CYP51 inhibitors.
Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development.
Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters.
Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.
Alkanes have been hypothesized to act as universal inducers of bacterial cytochrome P450 gene expression. We tested this hypothesis on an unusual P450 gene (cyp110) found on a conserved 11 kilobase episomal DNA element of unknown function found in filamentous cyanobacteria. We also monitored the binding of potential substrates to the P450 protein and explored the distribution of P450 protein in vegetative cells and nitrogen-fixing heterocysts using immuno-electron microscopy.
Hexadecane treatments resulted in a two-fold increase in mRNA, and a four-fold increase in P450 protein levels relative to control cultures. Hexane, octane and dodecane were toxic and induced substantial changes in membrane morphology. Long-chain saturated and unsaturated fatty acids were shown to bind the CYP110 protein using a spectroscopic spin-shift assay, but alkanes did not bind. CYP110 protein was detected in vegetative cells but not in differentiated heterocysts where nitrogen fixation occurs.
Hexadecane treatment was an effective inducer of CYP110 expression in cyanobacteria. Based on substrate binding profiles and amino acid sequence similarities it is hypothesized that CYP110 is a fatty acid ω-hydroxylase in photosynthetic cells. CYP110 was found associated with membrane fractions unlike other soluble microbial P450 proteins, and in this regard CYP110 more closely resembles eukarytotic P450s. Substrate stablization is an unlikely mechanism for alkane induction because alkanes did not bind to purified CYP110 protein.
Exposure to heat stress has been recognized as one of the major factors leading to the breakdown of the coral-alga symbiosis and coral bleaching. Here, we describe the presence of three new cytochrome P450 (CYP) genes from the reef-building coral endosymbiont Symbiodinium (type C3) and changes in their expression during exposure to severe and moderate heat stress conditions. Sequence analysis of the CYP C-terminal region and two conserved domains, the “PERF” and “heme-binding” domains, confirmed the separate identities of the CYP genes analyzed. In order to explore the effects of different heat stress scenarios, samples of the scleractinian coral Acropora millepora were exposed to elevated temperatures incrementally over an 18-h period (rapid thermal stress) and over a 120-h period (gradual thermal stress). After 18 h of gradual heating and incubation at 26°C, the Symbiodinium CYP mRNA pool was approximately 30% larger, while a further 6°C increase to a temperature above the average sea temperature (29°C after 72 h) resulted in a 2- to 4-fold increase in CYP expression. Both rapid heat stress and gradual heat stress at 32°C resulted in 50% to 90% decreases in CYP gene transcript abundance. Consequently, the initial upregulation of expression of CYP genes at moderately elevated temperatures (26°C and 29°C) was followed by a decrease in expression under the greater thermal stress conditions at 32°C. These findings indicate that in the coral-alga symbiosis under heat stress conditions there is production of chemical stressors and/or transcriptional factors that regulate the expression of genes, such as the genes encoding cytochrome P450 monooxygenases, that are involved in the first line of an organism's chemical defense.
Importance of the field
The cytochrome P450 (CYP) isoforms that are selectively induced following exposure to structurally-diverse chemicals often are the ones capable of metabolizing these chemicals. However, the molecular mechanism underlying this apparent functional coupling is not understood at present.
Areas covered in this review
Three hypotheses are developed to explain the complex process of selective chemical induction of CYPs: (i) each inducible CYP may have a corresponding intracellular receptor that interacts with the inducer chemical and mediates the selective induction of this CYP, (ii) each inducible CYP and its corresponding receptor may share a highly similar steric structure for their substrate/inducer binding sites, and (iii) each chemically-inducible CYP gene may have distinct genomic response element(s) that interact selectively with the corresponding receptor.
What the reader will gain
The readers are introduced to a novel theoretical framework that offers a plausible mechanistic explanation at the molecular levels concerning the complex process of how an organism selectively activates the biosynthesis of certain CYP isoform(s) that can effectively metabolize a chemical to which the organism is exposed.
Take home message
The theoretical framework developed herein seeks to ignite additional critical thinking on this important research subject as well as to promote experimental testing of the proposed theories in the future. Undoubtedly, these studies will enhance the understanding of the molecular mechanisms for the selective induction of CYP enzymes by chemicals.
Cytochrome P450 isoform; chemical inducers; selective enzyme induction; Ah receptor; nuclear receptors; mechanism of enzyme induction
The complete DNA sequence of Candida albicans DIT2, encoding cytochrome P450 family 56 (CYP56), was obtained, and heterologous expression was achieved in Escherichia coli, where CYP56 was targeted to the membrane fraction. In reconstituted assays with the purified enzyme, CYP56 was shown to catalyze the conversion of N-formyl tyrosine into N,N′-bisformyl dityrosine, a reaction that was dependent on cytochrome P450 reductase, NADPH, and oxygen, yielding a turnover of 21.6 min−1 and a ks of 26 μM. The Hill number was calculated as 1.6, indicating that two molecules of the substrate could bind to the protein. Azole antifungals could bind to the heme of CYP56 as a sixth ligand with high affinity. Both chromosomal alleles of CYP56 were disrupted using the SAT1 flipper technique, and CYP56 was found to be nonessential for cell viability under the culture conditions investigated. Susceptibility to azole drugs that bind to cytochromes P450 was tested, and the mutant showed unaltered susceptibility. However, the mutant showed increased susceptibility to the echinocandin drug caspofungin, suggesting an alteration in 1,3-glucan synthase and/or cell wall structure mediated by the presence of dityrosine. Phenotypically, the wild-type and mutant strains were morphologically similar when cultured in rich yeast extract-peptone-dextrose medium. However in minimal medium, the cyp56Δ mutant strain exhibited hyphal growth, in contrast to the wild-type strain, which grew solely in the yeast form. Furthermore, CYP56 was essential for chlamydospore formation.
Sterol 14α-demethylases (14DM) comprise the CYP51 cytochrome P450 genome family. The 14DM reaction is essential for the biosynthesis of sterols which are necessary for production of cellular membranes. This is the most widely distributed P450, being present in all biological kingdoms. From one kingdom to another the primary amino acid sequence identity usually ranges between 30-20%. In this minireview we describe the conservation of specific amino acids and the various CYP51 orthologs and indicate the roles that they may play in the structure/function of this monooxygenase. The prediction of the roles of different amino acids in 14DM is based on high resolution tertiary structures of these enzymes which set the stage for detailed understanding of the 14α-demethylase reaction and its selective, phyla-specific inhibition which is crucial for the design of potent inhibitors for treatment of infection by pathogenic microbes.
cytochrome P450; sterol 14α-demethylase; CYP51; crystal structure
The cytochrome P540 (CYP) superfamily currently includes about 9,000 proteins forming more than 800 families. The enzymes catalyze monooxygenation of a vast array of compounds and play essentially two roles. They provide biodefense (detoxification of xenobiotics, antibiotic production) and participate in biosynthesis of important endogenous molecules, particularly steroids. Based on these two roles, sterol 14|*alpha*|-demethylases (CYP51) belong to the second group of P450s. The CYP51 family, however, is very special as its members preserve strict functional conservation in enzyme activity in all biological kingdoms. At amino acid identity across the kingdoms as low as 25–30%, they all catalyze essentially the same three-step reaction of oxidative removal of the 14|*alpha*|-methyl group from the lanostane frame. This reaction is the required step in sterol biosynthesis of pathogenic microbes. We have shown that specific inhibition of protozoan CYP51 can potentially provide treatment for human trypanosomiases. Three sets of CYP51 inhibitors tested in vitro and in trypanosomal cells in this study include azoles [best results being 50% cell growth inhibition at <1 and at 1.3 µM for Trypanosoma cruzi (TC) and Trypanosoma brucei (TB), respectively], non-azole compounds (50% TC cell growth inhibition at 5 µM) and substrate analogs of the 14|*alpha*|-demethylase reaction. 32-Methylene cyclopropyl lanost-7-enol exhibited selectivity toward TC with 50% cell growth inhibition at 3 µM.
Cytochrome P450 (CYP51); Sterol biosynthesis; Sterol 14α-demethylase; Enzymatic activity; Inhibition; Sleeping sickness; Chagas disease; Antifungal agents; Antitrypanosomal drugs
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.
Recent studies have demonstrated that some morphologically atypical Aspergillus fumigatus strains are different species belonging to the section Fumigati. Aspergillus lentulus, one of these sibling species, is increasingly reported in patients under corticosteroid treatment. MICs of most antifungals in clinical use are elevated against A. lentulus, and it shows primary resistance to azole drugs. Two A. lentulus cytochrome P450 14-α sterol demethylases, encoded by A. lentulus cyp51A (Alcyp51A) and Alcyp51B genes, were identified. Targeted cyp51A gene knockout in A. lentulus showed that the intrinsic azole resistance of this species is cyp51A dependent. The Δcyp51A strain was morphologically indistinguishable from the A. lentulus wild-type strain, retaining the ability to cause pulmonary disease in neutropenic mice. The heterologous expression of A. lentulus cyp51A was performed in an A. fumigatus cyp51A-deficient strain, confirming that Cyp51A is responsible for the differences in A. lentulus-azole drug interaction.