Effects of Y132H and F145L substitutions on the activity, azole resistance and spectral properties of Candida albicans sterol 14-demethylase P450 (CYP51): a live example showing the selection of altered P450 through interaction with environmental compounds

Three variants of Candida albicans CYP51 (sterol 14-demethylase P450) having Y132H and/or F145L substitutions were purified and characterized to reveal the effects of these amino acid substitutions on the enzymatic properties and azole resistance of the enzyme. Y132H and F145L substitutions modified the spectral properties of the enzyme, suggesting that they caused some structural change modifying the heme environments of CYP51. Y132H and F145L substitutions increased the resistance of the enzyme to azole compounds but considerably decreased the catalytic activity. This fact represents a trade-off between acquisition of azole resistance and maintenance of high activity in the CYP51 having Y132H and F145L substitutions. A fluconazole-resistant C. albicans strain DUMC136 isolated from patients receiving long-term azole treatment was a homozygote of the altered CYP51 having Y132H and F145L substitutions. However, neither of these substitutions was found in CYP51 of wild-type C. albicans so far studied. These facts suggest that the azole-resistant variant having Y132H and/or F145L substitutions might be selected only under azole-rich environments because of its azole resistance and impaired catalytic activity. This may be a live example showing one of the important processes of P450 diversification, the selection of altered P450 through the interaction with environmental compounds.     

New Aspects on Lanosterol 14α-Demethylase and Cytochrome P450 Evolution: Lanosterol/Cycloartenol Diversification and Lateral Transfer

Sterol 14-demethylase (CYP51) is a member of the cytochrome P450 superfamily, widely found in animals, fungi, and plants but present in few prokaryotic groups. CYP51 is currently believed to be the ancestral cytochrome P450 that has been transferred from prokaryotes to eukaryotic kingdoms. We propose an alternate view of CYP51 evolution that has an impact on understanding the evolution of the entire CYP superfamily. Two hundred forty-nine bacterial and four archaeal CYP sequences have been aligned and a bacterial CYP tree designed, showing a separation of two branches. Prokaryotic CYP51s cluster to the minor branch, together with other eukaryote-like CYPs. Mycobacterial and methylococcal CYP51s cluster together (100% bootstrap probability), while Streptomyces CYP51 remains on a distant branch. A CYP51 phylogenetic tree has been constructed from 44 sequences resulting in a ((plant, bacteria),(animal, fungi)) topology (100% bootstrap probability). This is in accordance with the lanosterol/cycloartenol diversification of sterol biosynthesis. The lanosterol branch (nonphotosynthetic lineage) follows the previously proposed topology of animal and fungal orthologues (100% bootstrap probability), while plant and D. discoideum CYP51s belong to the cycloartenol branch (photosynthetic lineage), all in accordance with biochemical data. Bacterial CYP51s cluster within the cycloartenol branch (69% bootstrap probability), which is indicative of a lateral gene transfer of a plant CYP51 to the methylococcal/mycobacterial progenitor, suggesting further that bacterial CYP51s are not the oldest CYP genes. Lateral gene transfer is likely far more important than hitherto thought in the development of the diversified CYP superfamily. Consequently, bacterial CYPs may represent a mixture of genes with prokaryotic and eukaryotic origin.     

The preponderance of P450s in the Mycobacterium tuberculosis genome

The genome of Mycobacterium tuberculosis (Mtb) encodes 20 different cytochrome P450 enzymes (P450s). P450s are mono-oxygenases, which are historically considered to facilitate prokaryotic usage of unusual carbon sources. However, their preponderance in Mtb strongly indicates crucial physiological functions, as does the fact that polycyclic azoles (known P450 inhibitors) have potent anti-mycobacterial effects. Recent structural and enzyme characterization data reveal novel features for at least two Mtb P450s (CYP121 and CYP51). Genome analysis, knockout studies and structural comparisons signify important roles in cell biology and pathogenesis for various P450s and redox partner enzymes in Mtb. Elucidation of structure, function and metabolic roles will be essential in targeting the P450s as an 'Achilles heel' in this major human pathogen.     

Intron-exon organization and phylogeny in a large superfamily, the paralogous cytochrome P450 genes of Arabidopsis thaliana

The cytochrome P450 gene superfamily is represented by 80 genes in animal genomes and perhaps more than 300 genes in plant genomes. We analyzed about half of all Arabidopsis P450 genes, a very large dataset of truly paralogous genes. Sequence alignments were used to draw phylogenetic trees, and this information was compared with the intron-exon organization of each P450 gene. We found 60 unique intron positions, of which 37 were phase 0 introns. Our results confirm the polyphyletic origin of plant P450 genes. One group of these genes, the A-type P450s, are plant specific and characterized by a simple organization, with one highly conserved intron. Closely related A-type P450 genes are often clustered in the genome with as many as a dozen genes (e.g., of the CYP71 subfamily) on a short stretch of chromosome. The other P450 genes (non-A-type) form several distinct clades and are characterized by numerous introns. One such clade contains the two CYP51 genes, which are thought to encode obtusifoliol 14a demethylase. The two CYP51 genes have a single intron that is not shared with CYP51 genes from vertebrates or fungi, or with any other Arabidopsis P450 gene. Only a few of the Arabidopsis P450 genes are intronless (e.g., the CYP710A and CYP96A subfamilies). There was a relatively good correlation between intron conservation and phylogenetic relationships between members of the P450 subfamilies. Gene organization appears to be a useful tool in establishing the evolutionary relatedness of P450 genes, which may help in predictions of P450 function.     

Plant P450s as versatile drivers for evolution of species-specific chemical diversity

The irreversible nature of reactions catalysed by P450s makes these enzymes landmarks in the evolution of plant metabolic pathways. Founding members of P450 families are often associated with general (i.e. primary) metabolic pathways, restricted to single copy or very few representatives, indicative of purifying selection. Recruitment of those and subsequent blooms into multi-member gene families generates genetic raw material for functional diversification, which is an inherent characteristic of specialized (i.e. secondary) metabolism. However, a growing number of highly specialized P450s from not only the CYP71 clan indicate substantial contribution of convergent and divergent evolution to the observed general and specialized metabolite diversity. We will discuss examples of how the genetic and functional diversification of plant P450s drives chemical diversity in light of plant evolution. Even though it is difficult to predict the function or substrate of a P450 based on sequence similarity, grouping with a family or subfamily in phylogenetic trees can indicate association with metabolism of particular classes of compounds. Examples will be given that focus on multi-member gene families of P450s involved in the metabolic routes of four classes of specialized metabolites: cyanogenic glucosides, glucosinolates, mono- to triterpenoids and phenylpropanoids.     

Expression of human cytochromes P450 1A1 and P450 1A2 as fused enzymes with yeast NADPH-cytochrome P450 oxidoreductase in transgenic tobacco plants

Among 11 isoforms of the human cytochrome P450 enzymes metabolizing xenobiotics, CYP 1A1 and CYP 1A2 were major P450 species in the metabolism of the herbicides chlortoluron and atrazine in a yeast expression system. CYP1A2 was more active in the metabolism of both herbicides than CYP1A1. The fused enzymes of CYP1A1 and CYP1A2 with yeast NADPH-cytochrome P450 oxidoreductase were functionally active in the microsomal fraction of the yeast Saccharomyces cerevisiae and showed increased specific activity towards 7-ethoxyresorufin as compared to CYP1A1 and CYP1A2 alone. Then, both fused enzymes were each expressed in the microsomes of tobacco (Nicotiana tabacum cv. Samsun NN) plants. The transgenic plants expressing the CYP1A2 fusion enzyme had higher resistance to the herbicide chlortoluron than the plants expressing the CYP1A1 fusion enzyme did. The transgenic plants expressing the CYP1A2 fused enzyme metabolized chlortoluron to a larger extent to its non-phytotoxic metabolites through N-demethylation and ring-methyl hydroxylation as compared to the plants expressing the CYP1A1 fused enzyme. Thus, the possibility of increasing the herbicide resistance in the transgenic plants by the selection of P450 species and the fusion with P450 reductase is discussed.     

Arabidopsis CYP72C1 is an atypical cytochrome P450 that inactivates brassinosteroids

Cytochrome P450 monooxygenases (P450s) are a diverse family of proteins that have specialized roles in secondary metabolism and in normal cell development. Two P450s in particular, CYP734A1 and CYP72C1, have been identified as brassinosteroid-inactivating enzymes important for steroid-mediated signal transduction in Arabidopsis thaliana. Genetic analyses have demonstrated that these P450s modulate growth throughout plant development. While members of the CYP734A subfamily inactivate brassinosteroids through C-26 hydroxylation, the biochemical activity of CYP72C1 is unknown. Because CYP734A1 and CYP72C1 in Arabidopsis diverge more than brassinosteroid inactivating P450s in other plants, this study examines the structure and biochemistry of each enzyme. Three-dimensional models were generated to examine the substrate binding site structures and determine how they might affect the function of each P450. These models have indicated that the active site of CYP72C1 does not contain several conserved amino acids typically needed for substrate hydroxylation. Heterologous expression of these P450s followed by substrate binding analyses have indicated that CYP734A1 binds active brassinosteroids, brassinolide and castasterone, as well as their upstream precursors whereas CYP72C1 binds precursors more effectively. Seedling growth assays have demonstrated that the genetic state of CYP734A1, but not CYP72C1, affected responsiveness to high levels of exogenous brassinolide supporting our observations that CYP72C1 acts on brassinolide precursors. Although there may be some overlap in their physiological function, the distinct biochemical functions of these proteins in Arabidopsis has significant potential to fine-tune the levels of different brassinosteroid hormones throughout plant growth and development.     

Rapid P450 heme iron reduction by laser photoexcitation of Mycobacterium tuberculosis CYP121 and CYP51B1. Analysis of CO complexation reactions and reversibility of the P450/P420 equilibrium

We demonstrate that photoexcitation of NAD(P)H reduces heme iron of Mycobacterium tuberculosis P450s CYP121 and CYP51B1 on the microsecond time scale. Rates of formation for the ferrous-carbonmonoxy (Fe(II)-CO) complex were determined across a range of coenzyme/CO concentrations. CYP121 reaction transients were biphasic. A hyperbolic dependence on CO concentration was observed, consistent with the presence of a CO binding site in ferric CYP121. CYP51B1 absorption transients for Fe(II)-CO complex formation were monophasic. The reaction rate was second order with respect to [CO], suggesting the absence of a CO-binding site in ferric CYP51B1. In the absence of CO, heme iron reduction by photoexcited NAD(P)H is fast ( approximately 10,000-11,000 s(-1)) with both P450s. For CYP121, transients revealed initial production of the thiolate-coordinated (P450) complex (absorbance maximum at 448 nm), followed by a slower phase reporting partial conversion to the thiol-coordinated P420 species (at 420 nm). The slow phase amplitude increased at lower pH values, consistent with heme cysteinate protonation underlying the transition. Thus, CO binding occurs to the thiolate-coordinated ferrous form prior to cysteinate protonation. For CYP51B1, slow conversions of both the ferrous/Fe(II)-CO forms to species with spectral maxima at 423/421.5 nm occurred following photoexcitation in the absence/presence of CO. This reflected conversion from ferrous thiolate- to thiol-coordinated forms in both cases, indicating instability of the thiolate-coordinated ferrous CYP51B1. CYP121 Fe(II)-CO complex pH titrations revealed reversible spectral transitions between P450 and P420 forms. Our data provide strong evidence for P420 formation linked to reversible heme thiolate protonation, and demonstrate key differences in heme chemistry and CO binding for CYP121 and CYP51B1.     

Uncovering the role of hydrophobic residues in cytochrome P450-cytochrome P450 reductase interactions

Cytochrome P450 (CYP or P450)-mediated drug metabolism requires the interaction of P450s with their redox partner, cytochrome P450 reductase (CPR). In this work, we have investigated the role of P450 hydrophobic residues in complex formation with CPR and uncovered novel roles for the surface-exposed residues V267 and L270 of CYP2B4 in mediating CYP2B4--CPR interactions. Using a combination of fluorescence labeling and stopped-flow spectroscopy, we have investigated the basis for these interactions. Specifically, in order to study P450--CPR interactions, a single reactive cysteine was introduced in to a genetically engineered variant of CYP2B4 (C79SC152S) at each of seven strategically selected surface-exposed positions. Each of these cysteine residues was modified by reaction with fluorescein-5-maleimide (FM), and the CYP2B4-FM variants were then used to determine the K(d) of the complex by monitoring fluorescence enhancement in the presence of CPR. Furthermore, the intrinsic K(m) values of the CYP2B4 variants for CPR were measured, and stopped-flow spectroscopy was used to determine the intrinsic kinetics and the extent of reduction of the ferric P450 mutants to the ferrous P450--CO adduct by CPR. A comparison of the results from these three approaches reveals that the sites on P450 exhibiting the greatest changes in fluorescence intensity upon binding CPR are associated with the greatest increases in the K(m) values of the P450 variants for CPR and with the greatest decreases in the rates and extents of reduced P450--CO formation.     

Crystal structure of the Mycobacterium tuberculosis P450 CYP121-fluconazole complex reveals new azole drug-P450 binding mode

Azole and triazole drugs are cytochrome P450 inhibitors widely used as fungal antibiotics and possessing potent antimycobacterial activity. We present here the crystal structure of Mycobacterium tuberculosis cytochrome P450 CYP121 in complex with the triazole drug fluconazole, revealing a new azole heme ligation mode. In contrast to other structurally characterized cytochrome P450 azole complexes, where the azole nitrogen directly coordinates the heme iron, in CYP121 fluconazole does not displace the aqua sixth heme ligand but occupies a position that allows formation of a direct hydrogen bond to the aqua sixth heme ligand. Direct ligation of fluconazole to the heme iron is observed in a minority of CYP121 molecules, albeit with severe deviations from ideal geometry due to close contacts with active site residues. Analysis of both ligand-on and -off structures reveals the relative position of active site residues derived from the I-helix is a key determinant in the relative ratio of on and off states. Regardless, both ligand-bound states lead to P450 inactivation by active site occlusion. This previously unrecognized means of P450 inactivation is consistent with spectroscopic analyses in both solution and in the crystalline form and raises important questions relating to interaction of azoles with both pathogen and human P450s.     

Modelling of three-dimensional structures of cytochromes P450 11B1 and 11B2.

The final steps of the biosynthesis of glucocorticoids and mineralocorticoids in the adrenal cortex require the action of two different cytochromes P450--CYP11B1 and CYP11B2. Homology modelling of the three-dimensional structures of these cytochromes was performed based on crystallographic coordinates of two bacterial P450s, CYP102 (P450BM-3) and CYP108 (P450terp). Principal attention was given to the modelling of the active sites and a comparison of the active site structures of CYP11B1 and CYP11B2 was performed. It can be demonstrated that key residue contacts within the active site appear to depend on the orientation of the heme. The obtained 3D structures of CYP11B1 and CYP11B2 were used for investigation of structure-function relationships of these enzymes. Previously obtained results on naturally occurring mutants and on mutants obtained by site-directed mutagenesis are discussed.     

Differences in flexibility of active sites of cytochromes P450 probed by resonance Raman and UV-Vis absorption spectroscopy.

Spectroscopic methods reveal differences in flexibility and stability of P450 forms. Among microsomal P450s, the most flexible active site has been found in the CYP3A4 enzyme as it is compressible and the heme vinyl side chains may adopt two different conformations. On the other hand, active site of this enzyme denatures quite easily upon hydrostatic pressure. The most rigid active site able to withstand the effect of high pressure has CYP1A2. The bacterial CYP102 (BM3) flavocytochrome has also a rather stable, but flexible active site. The differences between CYP3A4 and CYP1A2 active sites apparently reflect their ability to bind various substrates: whereas the CYP3A4 binds a vast variety of structures, the CYP1A2 preferentially binds planar, aromatic structures and its substrate specificity is relatively narrow.     

Conversion and synthesis of chemicals catalyzed by fungal cytochrome P450 monooxygenases: A review

Cytochrome P450s (also called CYPs or P450s) are a superfamily of heme-containing monooxygenases. They are distributed in all biological kingdoms. Most fungi have at least two P450-encoding genes, CYP51 and CYP61, which are housekeeping genes that play important roles in the synthesis of sterols. However, the kingdom fungi is an interesting source of numerous P450s. Here, we review reports on fungal P450s and their applications in the bioconversion and biosynthesis of chemicals. We highlight their history, availability, and versatility. We describe their involvement in hydroxylation, dealkylation, oxygenation, C═C epoxidation, C–C cleavage, C–C ring formation and expansion, C–C ring contraction, and uncommon reactions in bioconversion and/or biosynthesis pathways. The ability of P450s to catalyze these reactions makes them promising enzymes for many applications. Thus, we also discuss future prospects in this field. We hope that this review will stimulate further study and exploitation of fungal P450s for specific reactions and applications.     

Phylogenetic and Functional Analysis of the Vertebrate Cytochrome P450 2 Family

Cytochrome P450 (CYP) proteins compose a highly diverse superfamily found in all domains of life. These proteins are enzymes involved in metabolism of endogenous and exogenous compounds. In vertebrates, the CYP2 family is one of the largest, most diverse and plays an important role in mammalian drug metabolism. However, there are more than 20 vertebrate CYP2 subfamilies with uncertain evolution and fairly discrete subfamily composition within vertebrate classes, hindering extrapolation of knowledge across subfamilies. To better understand CYP2 diversity, a phylogenetic analysis of 196 CYP2 protein sequences from 16 species was performed using a maximum likelihood approach and Bayesian inference. The analyses included the CYP2 compliment from human, fugu, zebrafish, stickleback, medaka, cow, and dog genomes. Additional sequences were included from rabbit, marsupial, platypus, chicken, frog, and salmonid species. Three CYP2 sequences from the tunicate Ciona intestinalis were utilized as the outgroup. Results indicate a single ancestral vertebrate CYP2 gene and monophyly of all CYP2 subfamilies. Two subfamilies (CYP2R and CYP2U) pre-date vertebrate diversification, allowing direct comparison across vertebrate classes, while all other subfamilies originated during vertebrate diversification, often within specific vertebrate lineages. Analysis of site-specific evolution indicates that some substrate recognition sites (SRS) previously proposed for CYP genes do not have elevated rates of evolution, suggesting that these regions of the protein are not necessarily important in recognition of CYP2 substrates. Type II functional divergence analysis identified multiple residues in the active site of CYP2F, CYP2A, and CYP2B proteins that have undergone radical biochemical changes and may be functionally important.     

Plant NADPH-cytochrome P450 oxidoreductases

NADPH-cytochrome P450 oxidoreductase (CPR) serves as the electron donor to almost all eukaryotic cytochromes P450. It belongs to a small family of diflavin proteins and is built of cofactor binding domains with high structural homology to those of bacterial flavodoxins and to ferredoxin-NADP⁺ oxidoreductases. CPR shuttles electrons from NADPH through the FAD and FMN-cofactors into the central heme-group of the P450s. Mobile domains in CPR are essential for electron transfer between FAD and FMN and for P450 interaction. Blast searches identified 54 full-length gene sequences encoding CPR derived from a total of 35 different plant species. CPRs from vascular plants cluster into two major phylogenetic groups. Depending on the species, plants contain one, two or three paralogs of which one is inducible. The nature of the CPR–P450 interacting domains is well conserved as demonstrated by the ability of CPRs from different species or even from different kingdoms to at least partially complement each other functionally. This makes CPR an ideal bio-brick in synthetic biology approaches to re-design or develop entirely different combinations of existing biological systems to gain improved or completely altered functionalities based on the “share your parts” principle.     

In silico analysis of cytochrome P450 monooxygenases in chronic granulomatous infectious fungus Sporothrix schenckii: Special focus on CYP51

Sporotrichosis is an emerging chronic, granulomatous, subcutaneous, mycotic infection caused by Sporothrix species. Sporotrichosis is treated with the azole drug itraconazole as ketoconazole is ineffective. It is a well-known fact that azole drugs act by inhibiting cytochrome P450 monooxygenases (P450s), heme-thiolate proteins. To date, nothing is known about P450s in Sporothrix schenckii and the molecular basis of its resistance to ketoconazole. Here we present genome-wide identification, annotation, phylogenetic analysis and comprehensive P450 family-level comparative analysis of S. schenckii P450s with pathogenic fungi P450s, along with a rationale for ketoconazole resistance by S. schenckii based on in silico structural analysis of CYP51. Genome data-mining of S. schenckii revealed 40 P450s in its genome that can be grouped into 32 P450 families and 39 P450 subfamilies. Comprehensive comparative analysis of P450s revealed that S. schenckii shares 11 P450 families with plant pathogenic fungi and has three unique P450 families: CYP5077, CYP5386 and CYP5696 (novel family). Among P450s, CYP51, the main target of azole drugs was also found in S. schenckii. 3D modeling of S. schenckii CYP51 revealed the presence of characteristic P450 motifs with exceptionally large reductase interaction site 2. In silico analysis revealed number of mutations that can be associated with ketoconazole resistance, especially at the channel entrance to the active site. One of possible reason for better stabilization of itraconazole, compared to ketoconazole, is that the more extended molecule of itraconazole may form a hydrogen bond with ASN-230. This in turn may explain its effectiveness against S. schenckii vis-a-vis resistant to ketoconazole. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

Molecular identification and functional characterization of cytochrome P450 monooxygenases from the brown-rot basidiomycete Postia placenta

We explored the molecular diversity and functional capabilities of cytochrome P450 monooxygenases (P450s) from the brown-rot basidiomycete Postia placenta. Using bioinformatic and experimental data, we found 250 genes of P450s in the whole genome, including 60 putative allelic variants. Phylogenetic analysis revealed the presence of 42 families, including 18 novel families. Comparative phylogenetic analysis of P450s from P. placenta and the white-rot basidiomycete Phanerochaete chrysosporium suggested that vigorous gene duplication and molecular evolution occurred after speciation of basidiomycetes. Among the 250 gene models, 184 were isolated as full-length cDNA and transformed into Saccharomyces cerevisiae to construct a functional library in which recombinant P450s were co-expressed with yeast NADPH-P450 oxidoreductase. Using this library, the catalytic potentials of P450s against a wide variety of compounds were investigated. A functionomic survey allowed the discovery of novel catalytic properties of P. placenta P450s. The phylogenetic diversity of the CYP53 family in P. placenta was clear, and CYP53D2 is capable of converting stilbene derivatives. This is the first report of this peculiar function of the CYP53 family. Our increased understanding of the molecular and functional diversity of P450s in this fungus will facilitate comprehension of metabolic diversity in basidiomycetes and has future biotechnology applications.     

Flexibility and stability of the structure of cytochromes P450 3A4 and BM-3.

The flexibility of the structure and compressibility of the respective active site of cytochromes P450 3A4 (CYP3A4) and BM-3 (CYP102) were studied using absorption spectroscopy in the ultraviolet and visual regions. Conformational changes in the overall protein structures of both CYP3A4 and CYP102 due to the effects of temperature and pressure are reversible. However, the enzymes differ in the properties of their active sites. The CYP3A4 enzyme denatures to the inactive P420 form relatively easy, at 3000 bar over half is converted to P420. The compressibility of its active site is lower than that of CYP102 and is greater with the substrate bound, which is in line with the observed lack of a stabilizing effect of the substrate on its conformation under pressure. In contrast, CYP102, although having the most compressible active site among the P450s, possesses a structure that does not denature easily to the inactive (P420) form under pressure. In this respect, it resembles the P450 isolated from acidothermophilic archaebacteria [McLean, M.A., Maves, S.A., Weiss, K.E., Krepich, S. & Sligar, S.G. (1998) Biochem. Biophys. Res. Commun. 252, 166-172].     

Targeting of the highly conserved threonine 302 residue of cytochromes P450 2B family during mechanism-based inactivation by aryl acetylenes

Cytochromes P450 (CYPs or P450s) contain a highly conserved threonine residue in the active site, which is referred to as Thr302 in the amino acid sequence of CYP2B4. Extensive biochemical and crystallographic studies have established that this Thr302 plays a critical role in activating molecular oxygen to generate Compound I, a putative iron(IV)-oxo porphyrin cation radical, that carries out the preliminary oxygenation of CYP substrates. Because of its proximity to the center of the P450 active site, this Thr302 is susceptible to mechanism-based inactivation under certain conditions. In this article, we review recent studies on the mechanism-based inactivation of three mammalian P450s in the 2B family, CYP2B1 (rat), 2B4 (rabbit) and 2B6 (human) by tert-butylphenylacetylene (tBPA). These studies showed that tBPA is a potent mechanism-based inactivator of CYP2B1, 2B4 and 2B6 with high k_inact/K_I ratios (0.23–2.3 min⁻¹ μM⁻¹) and low partition ratios (0–5). Furthermore, mechanistic studies revealed that tBPA inactivates these three CYP2B enzymes through the formation of a single ester adduct with the Thr302 in the active site. These inhibitory properties of tBPA allowed the preparation of a modified CYP2B4 where the Thr302 was covalently and stoichiometrically labeled by a reactive intermediate of tBPA in quantities large enough to permit spectroscopic and crystallographic studies of the consequences of covalent modification of Thr302. Molecular modeling studies revealed a unique binding mode of tBPA in the active site that may shed light on the potency of this inhibition. The results from these studies may serve as a basis for designing more specific and potent inhibitors for P450s by targeting this highly conserved threonine residue which is present in the active sites of most mammalian P450s.     

Biophysical characterization of the sterol demethylase P450 from Mycobacterium tuberculosis, its cognate ferredoxin, and their interactions

Mycobacterium tuberculosis encodes a P450 of the sterol demethylase family (CYP51) chromosomally located adjacent to a ferredoxin (Fdx). CYP51 and Fdx were purified to homogeneity and characterized. Spectroscopic analyses were consistent with cysteinate- and aqua-ligated heme iron in CYP51. An epsilon419 of 134 mM(-1) cm(-1) was determined for oxidized CYP51. Analysis of interactions of 1-, 2-, and 4-phenylimidazoles with CYP51 showed that the 1- and 4-forms were heme iron-coordinating inhibitors, while 2-phenylimidazole induced a substrate-like optical shift. The 2-phenyimidazole-bound CYP51 demonstrated unusual decreases in high-spin heme iron content at elevated temperatures and an almost complete absence of high-spin heme iron by low-temperature EPR. These data suggest thermally induced alterations in CYP51 active site structure and/or binding modes for the small ligand. Reduction of CYP51 in the presence of carbon monoxide leads to formation of an Fe(II)-CO complex with a Soret absorption maximum at 448.5 nm, which collapses (at 0.246 min(-1) at pH 7.0) forming a species with a Soret maximum at 421.5 nm (the inactive P420 form). The rate of P420 formation is accelerated at lower pH, consistent with protonation of the cysteinate (Cys 394) to a thiol underlying the P450-P420 transition. The P450 form is stabilized by estriol, which induces a type I spectral shift on binding CYP51 (Kd = 21.7 microM). Nonstandard spectral changes occur on CYP51 reduction (using either dithionite or natural redox partners), including a blue-shifted Soret band and development of a strong feature at approximately 558.5 nm, suggestive of cysteine thiol ligation. Thus, ligand-free ferrous CYP51 is prone to thiolate ligand protonation even in the absence of carbon monoxide. Analysis of reoxidized CYP51 demonstrates that the enzyme re-forms P450, indicating that Cys 394 thiol is readily deprotonated to thiolate in the ferric form. Spectroscopic analysis of Fdx by EPR (resonance at g = 2.03) and magnetic CD (intensity for oxidized and reduced forms and signal intensity dependence on field strength and temperature) demonstrated that Fdx binds a [3Fe-4S] iron-sulfur cluster. Potentiometric studies show that the midpoint potential for ligand-free CYP51 is -375 mV, increasing to -225 mV in the estriol-bound form. The Fdx potential is -31 mV. Fdx forms a productive electron transfer complex with CYP51 and reduces it at a rate of 3.0 min(-1) in the ligand-free form and 4.3 min(-1) in the estriol-bound form, despite a thermodynamic barrier. Steady-state analysis of a M. tuberculosis class I redox system comprising flavoprotein reductase A (FprA), Fdx, and estriol-bound CYP51 indicates heme iron reduction as a rate-limiting step.