Impact of recently emerged sterol 14{alpha}-demethylase (CYP51) variants of Mycosphaerella graminicola on azole fungicide sensitivity

The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14α-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.     

Evolution of the CYP51 gene in Mycosphaerella graminicola: evidence for intragenic recombination and selective replacement

Recent findings are consistent with a slow but constant shift towards reduced sensitivity of Mycosphaerella graminicola to azole fungicides, which target the CYP51 gene. The goal of this study was to elucidate the evolutionary mechanisms through which CYP51-based mutations associated with altered sensitivity have evolved in M. graminicola over space and time. To accomplish this, we sequenced and compared a portion of the CYP51 gene encompassing the main mutations associated with altered sensitivity towards demethylation inhibitor fungicides. The CYP51 gene showed an extraordinary dynamic shift consistent with a selective haplotype replacement both in space and in time. No mutations associated with increased resistance to azoles were found in non-European populations. These mutations were also absent in the oldest collections from Europe, whereas they dominated in the recent European populations. Intragenic recombination was identified as an important evolutionary process in populations affected by high fungicide selection, suggesting the creation of novel alleles among existing mutations as a potential source of novel resistance alleles. We propose that CYP51 mutations giving resistance in M. graminicola arose only locally (perhaps in Denmark or the UK) and were then spread eastward across Europe through wind-dispersed ascospores. We conclude that recurring cycles of recombination coupled with selection due to the widespread use of azole fungicides will increase the frequency of novel mutants or recombinants with higher resistance. Long-distance gene flow due to wind dispersal of ascospores will move the resulting new alleles to new areas following the prevailing wind directions. A selective replacement favouring haplotypes with various coding mutations at the target site for azole fungicides during the last 5-10 years is the most likely cause of the decrease in sensitivity reported for many azole fungicides in the same period.     

Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase

Mycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14α-demethylase (CYP51), which requires cytochrome P450 reductase (CPR) as its redox partner for functional activity. The M. graminicola CPR (MgCPR) is able to catalyze the sterol 14α-demethylation of eburicol and lanosterol when partnered with Candida albicans CYP51 (CaCYP51) and that of eburicol only with M. graminicola CYP51 (MgCYP51). The availability of the functional in vivo redox partner enabled the in vitro catalytic activity of MgCYP51 to be demonstrated for the first time. MgCYP51 50% inhibitory concentration (IC₅₀) studies with epoxiconazole, tebuconazole, triadimenol, and prothioconazole-desthio confirmed that MgCYP51 bound these azole inhibitors tightly. The characterization of the MgCPR/MgCYP51 redox pairing has produced a functional method to evaluate the effects of agricultural azole fungicides, has demonstrated eburicol specificity in the activity observed, and supports the conclusion that prothioconazole is a profungicide.     

A novel substitution I381V in the sterol 14α-demethylase (CYP51) of Mycosphaerella graminicola is differentially selected by azole fungicides

The recent reduction in the efficacy of azole fungicides in controlling Septoria leaf blotch of wheat, caused by Mycosphaerella graminicola , has prompted concerns over possible development of resistance, particularly in light of the recent emergence of widespread resistance to quinone outside inhibitors (QoIs). We have recently implicated alterations in the target-encoding sterol 14α-demethylase protein (CYP51), and over-expression of genes encoding efflux pumps, in reducing sensitivity to the azole class of sterol demethylation inhibitors (DMIs) in M. graminicola . Here we report on the prevalence and selection of two CYP51 alterations, substitution I381V and deletion of codons 459 and 460 (ΔY459/G460), in populations of M .  graminicola . Neither alteration has previously been identified in human or plant pathogenic fungi resistant to azoles. The presence of ΔY459/G460 showed a continuous distribution of EC₅₀ values across isolates with either I381 or V381, and had no measurable effect on azole sensitivity. Data linking fungicide sensitivity with the presence of I381V in M. graminicola show for the first time that a particular CYP51 alteration is differentially selected by different azoles in field populations of a plant pathogen. Substitution I381V although not an absolute requirement for reduced azole sensitivity, is selected by tebuconazole and difenoconazole treatment, suggesting an adaptive advantage in the presence of these two compounds. Prochloraz treatments appeared to select negatively for I381V, whereas other azole treatments did not or only weakly impacted on the prevalence of this substitution. These findings suggest treatments with different members of the azole class of fungicides could offer a resistance management strategy.     

Cloning of the CYP51 gene from the eyespot pathogen Tapesia yallundae indicates that resistance to the DMI fungicide prochloraz is not related to sequence changes in the gene encoding the target site enzyme

Resistance to sterol 14 alpha-demethylase inhibitor (DMI) fungicides has been correlated with mutations in the CYP51 gene encoding the target enzyme eburicol 14 alpha-demethylase. CYP51 was isolated from the eyespot pathogen Tapesia yallundae revealing a predicted 526-amino acid product exhibiting homology to other fungal CYP51s. CYP51 was sequenced from four field isolates sensitive or resistant to the DMI fungicide prochloraz and partially sequenced from two further isolates and eight progeny from a cross between prochloraz-sensitive and -resistant parents. Two alleles of the gene were detected termed CYP51-1 and CYP51-2. No correlation was found between sequence change and fungicide sensitivity. Therefore prochloraz resistance involved a mechanism other than mutation in the target site gene.     

Sequence variation in the CYP51 gene of Blumeria graminis associated with resistance to sterol demethylase inhibiting fungicides

Resistance to sterol 14alpha-demethylase inhibiting fungicides (DMIs) has been correlated with mutations in the CYP51 gene, which encodes the target enzyme eburicol 14alpha-demethylase. To test the hypothesis that variation in the CYP51 gene explains variation for DMI sensitivity in barley and wheat powdery mildew species, this gene was sequenced from isolates of Blumeria graminis f.sp. hordei (Bgh) and f.sp. tritici (Bgt), respectively, which differed in their responses to DMIs in agricultural populations in the UK. Two single-nucleotide mutations in the CYP51 gene, which resulted in the amino acid substitutions Y136F and K147Q, were detected. K147Q is a novel mutation present only in Bgh isolates expressing very high levels of resistance. Sequence analysis of the CYP51 gene from the progeny of a cross between DMI-sensitive and resistant Bgh isolates showed that both mutations segregate with resistance, which is consistent with CYP51 controlling a major portion of DMI resistance. However, genetic analysis of resistance to the DMI triadimenol indicates that mutation of the CYP51 gene is not the only mechanism of resistance operating in B. graminis.     

Mechanism of binding of prothioconazole to Mycosphaerella graminicola CYP51 differs from that of other azole antifungals

Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 μg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml(-1) resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.     

Estriol bound and ligand-free structures of sterol 14alpha-demethylase

Sterol 14alpha-demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes and drug targets in antifungal therapy. Here, we report CYP51 structures in ligand-free and estriol bound forms. Using estriol as a probe, we determined orientation of the substrate in the active site, elucidated protein contacts with the invariant 3beta-hydroxy group of a sterol, and identified F78 as a key discriminator between 4alpha-methylated and 4alpha,beta-dimethylated substrates. Analysis of CYP51 dynamics revealed that the C helix undergoes helix-coil transition upon binding and dissociation of a ligand. Loss of helical structure of the C helix in the ligand-free form results in an unprecedented opening of the substrate binding site. Upon binding of estriol, the BC loop loses contacts with molecular surface and tends to adopt a closed conformation. A mechanism for azole resistance in the yeast pathogen Candida albicans associated with mutations in the ERG11 gene encoding CYP51 is suggested based on CYP51 protein dynamics.     

Optimized expression and catalytic properties of a wheat obtusifoliol 14alpha-demethylase (CYP51) expressed in yeast. Complementation of erg11Delta yeast mutants by plant CYP51.

CYP51s form the only family of P450 proteins conserved in evolution from prokaryotes to fungi, plants and mammals. In all eukaryotes, CYP51s catalyse 14alpha-demethylation of sterols. We have recently isolated two CYP51 cDNAs from sorghum [Bak, S., Kahn, R.A., Olsen, C. E. & Halkier, B.A. (1997) Plant J. 11, 191-201] and wheat [Cabello-Hurtado, F., Zimmerlin, A., Rahier, A., Taton, M., DeRose, R., Nedelkina, S., Batard, Y., Durst, F., Pallett, K.E. & Werck-Reichhart, D. (1997) Biophys. Biochem. Res. Commun. 230, 381-385]. Wheat and sorghum CYP51 proteins show a high identity (92%) compared with their identity with their fungal and mammalian orthologues (32-39%). Data obtained with plant microsomes have previously suggested that differences in primary sequences reflect differences in sterol pathways and CYP51 substrate specificities between animals, fungi and plants. To investigate more thoroughly the properties of the plant CYP51, the wheat enzyme was expressed in yeast strains overexpressing different P450 reductases as a fusion with either yeast or plant (sorghum) membrane targeting sequences. The endogenous sterol demethylase gene (ERG11) was then disrupted. A sorghum-wheat fusion protein expressed with the Arabidopsis thaliana reductase ATR1 showed the highest level of expression and activity. The expression induced a marked proliferation of microsomal membranes so as to obtain 70 nmol P450.(L culture)-1, with CYP51 representing 1.5% of microsomal protein. Without disruption of the ERG11 gene, the expression level was fivefold reduced. CYP51 from wheat complemented the ERG11 disruption, as the modified yeasts did not need supplementation with exogenous ergosterol and grew normally under aerobic conditions. The fusion plant enzyme catalysed 14alpha-demethylation of obtusifoliol very actively (Km,app = 197 microm, kcat = 1.2 min-1) and with very strict substrate specificity. No metabolism of lanosterol and eburicol, the substrates of the fungal and mammalian CYP51s, nor metabolism of herbicides and fatty acids was detected in the recombinant yeast microsomes. Surprisingly lanosterol (Ks = 2.2 microM) and eburicol (Ks = 2.5 microm) were found to bind the active site of the plant enzyme with affinities higher than that for obtusifoliol (Ks = 289 microM), giving typical type-I spectra. The amplitudes of these spectra, however, suggested that lanosterol and eburicol were less favourably positioned to be metabolized than obtusifoliol. The recombinant enzyme was also used to test the relative binding constants of two azole compounds, LAB170250F and gamma-ketotriazole, which were previously reported to be potent inhibitors of the plant enzyme. The Ks of plant CYP51 for LAB170250F (0.29 microM) and gamma-ketotriazole (0.40 microM) calculated from the type-II sp2 nitrogen-binding spectra were in better agreement with their reported effects as plant CYP51 inhibitors than values previously determined with plant microsomes. This optimized expression system thus provides an excellent tool for detailed enzymological and mechanistic studies, and for improving the selectivity of inhibitory molecules.     

Prothioconazole and Prothioconazole-Desthio Activities against Candida albicans Sterol 14-α-Demethylase

Prothioconazole is a new triazolinthione fungicide used in agriculture. We have used Candida albicans CYP51 (CaCYP51) to investigate the in vitro activity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment of C. albicans cells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts from C. albicans grown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.     

CYP51 from Trypanosoma brucei is obtusifoliol-specific

New isoforms of CYP51 (sterol 14alpha-demethylase), an essential enzyme in sterol biosynthesis and primary target of azole antimycotic drugs, are found in pathogenic protists, Trypanosoma brucei(TB), T. vivax, T. cruzi, and Leishmania major. The sequences share approximately 80% amino acid identity and are approximately 25% identical to sterol 14alpha-demethylases from other biological kingdoms. Differences of residues conserved throughout the rest of the CYP51 family that align with the BC-loop and helices F and G of CYP51 from Mycobacterium tuberculosis (MT)) imply possible alterations in the topology of the active site cavity of the protozoan enzymes. CYP51 and cytochrome P450 reductase (CPR) from TB were cloned, expressed in Escherichia coli, and purified. The P450 has normal spectral features (including absolute absorbance, carbon monoxide, and ligand binding spectra), is efficiently reduced by TB and rat CPR but demonstrates altered specificity in comparison with human CYP51 toward three tested azole inhibitors, and contrary to the human, Candida albicans, and MT isoforms, reveals profound substrate preference toward obtusifoliol (turnover 5.6 min(-1)). It weakly interacts with the other known CYP51 substrates; slow lanosterol conversion predominantly produces the 14alpha-carboxyaldehyde intermediate. Although obtusifoliol specificity is typical for plant isoforms of CYP51, the set of sterol biosynthetic enzymes in the protozoan genomes together with available information about sterol composition of kinetoplastid cells suggest that the substrate preference of TBCYP51 may reflect a novel sterol biosynthetic pathway in Trypanosomatidae.     

Plant sterol 14 alpha-demethylase affinity for azole fungicides

Azole fungicides were thought to have much greater affinity for the fungal cytochrome P450 enzyme, sterol 14 alpha-demthylase (CYP51) than the plant orthologue. Using purified CYP51 from the plant Sorghum bicolor L Moenech, a direct comparison of the sensitivity to the fungicides triadimenol and tebuconazole has been carried out. S. bicolor CYP51 was purified to homogenity as determined by SDS--PAGE and specific heme content. Addition of the azole fungicides triadimenol and tebuconazole induced type II spectral changes, with saturation occurring at equimolar azole/P450 concentrations. Inhibition of reconstituted activities revealed only a threefold insensitivity of the plant CYP51 compared to a fungal CYP51, from the phytopathogen Ustilago maydis, as judged by IC(50) values. The implications for fungicide mode of action and application are discussed.     

Molecular modelling of the emergence of azole resistance in Mycosphaerella graminicola

A structural rationale for recent emergence of azole (imidazole and triazole) resistance associated with CYP51 mutations in the wheat pathogen Mycosphaerella graminicola is presented, attained by homology modelling of the wild type protein and 13 variant proteins. The novel molecular models of M. graminicola CYP51 are based on multiple homologues, individually identified for each variant, rather than using a single structural scaffold, providing a robust structure-function rationale for the binding of azoles, including important fungal specific regions for which no structural information is available. The wild type binding pocket reveals specific residues in close proximity to the bound azole molecules that are subject to alteration in the variants. This implicates azole ligands as important agents exerting selection on specific regions bordering the pocket, that become the focus of genetic mutation events, leading to reduced sensitivity to that group of related compounds. Collectively, the models account for several observed functional effects of specific alterations, including loss of triadimenol sensitivity in the Y137F variant, lower sensitivity to tebuconazole of I381V variants and increased resistance to prochloraz of V136A variants. Deletion of Y459 and G460, which brings about removal of that entire section of beta turn from the vicinity of the binding pocket, confers resistance to tebuconazole and epoxiconazole, but sensitivity to prochloraz in variants carrying a combination of A379G I381V ΔY459/G460. Measurements of binding pocket volume proved useful in assessment of scope for general resistance to azoles by virtue of their accommodation without bonding interaction, particularly when combined with analysis of change in positions of key amino acids. It is possible to predict the likely binding orientation of an azole molecule in any of the variant CYPs, providing potential for an in silico screening system and reliable predictive approach to assess the probability of particular variants exhibiting resistance to particular azole fungicides.     

Heterologous expression and characterization of the sterol 14α-demethylase CYP51F1 from Candida albicans

Lanosterol 14α-demethylase (CYP51F1) from Candida albicans is known to be an essential enzyme in fungal sterol biosynthesis. Wild-type CYP51F1 and several of its mutants were heterologously expressed in Escherichia coli, purified, and characterized. It exhibited a typical reduced CO-difference spectrum with a maximum at 446 nm. Reconstitution of CYP51F1 with NADPH-P450 reductase gave a system that successfully converted lanosterol to its demethylated product. Titration of the purified enzyme with lanosterol produced a typical type I spectral change with K_d = 6.7 μM. The azole antifungal agents econazole, fluconazole, ketoconazole, and itraconazole bound tightly to CYP51F1 with K_d values between 0.06 and 0.42 μM. The CYP51F1 mutations F105L, D116E, Y132H, and R467K frequently identified in clinical isolates were examined to determine their effect on azole drug binding affinity. The azole K_d values of the purified F105L, D116E, and R467K mutants were little altered. A homology model of C. albicans CYP51F1 suggested that Tyr132 in the BC loop is located close to the heme in the active site, providing a rationale for the modified heme environment caused by the Y132H substitution. Taken together, functional expression and characterization of CYP51F1 provide a starting basis for the design of agents effective against C. albicans infections.     

Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (K_s, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (K_d, ≤3 nM) and prothioconazole-desthio the lowest (K_d, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC₅₀s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC₁₀₀, >256 μg ml⁻¹). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC₁₀₀ [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml⁻¹) with the exception of clotrimazole, which was as potent as malachite green (MIC₁₀₀, ∼1 μg ml⁻¹). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC₅₀, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC₁₀₀, ∼1 to 2 μg ml⁻¹) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.     

Substrate recognition sites in 14alpha-sterol demethylase from comparative analysis of amino acid sequences and X-ray structure of Mycobacterium tuberculosis CYP51.

The crystal structure of 14alpha-sterol demethylase from Mycobacterium tuberculosis (MTCYP51) [Proc. Natl. Acad. Sci. USA 98 (2001) 3068-3073] provides a template for analysis of eukaryotic orthologs which constitute the CYP51 family of cytochrome P450 proteins. Putative substrate recognition sites (SRSs) were identified in MTCYP51 based on the X-ray structures and have been compared with SRSs predicted based on Gotoh's analysis [J. Biol. Chem. 267 (1992) 83-90]. While Gotoh's SRS-4, 5, and 6 contribute in formation of the putative MTCYP51 substrate binding site, SRS-2 and 3 likely do not exist in MTCYP51. SRS-1, as part of the open BC loop, in the conformation found in the crystal can provide only limited contacts with the sterol. However, its role in substrate binding might dramatically increase if the loop closes in response to substrate binding. Thus, while the notion of SRSs has been very useful in leading to our current understanding of P450 structure and function, their identification by sequence alignment between distant P450 families will not necessarily be a good predictor of residues associated with substrate binding. Localization of CYP51 mutation hotspots in Candida albicans azole resistant isolates was analyzed with respect to SRSs. These mutations are found to be outside of the putative substrate interacting sites indicating the preservation of the protein active site under the pressure of azole treatment. Since the mutations residing outside the putative CYP51 active side can profoundly influence ligand binding within the active site, perhaps they provide insight into the basis of evolutionary changes which have occurred leading to different P450s.     

Conservation in the CYP51 family. Role of the B' helix/BC loop and helices F and G in enzymatic function

CYP51 (sterol 14 alpha-demethylase) is an essential enzyme in sterol biosynthetic pathways and the only P450 gene family having catalytically identical orthologues in different biological kingdoms. The proteins have low sequence similarity across phyla, and the whole family contains about 40 completely conserved amino acid residues. Fifteen of these residues lie in the secondary structural elements predicted to form potential substrate recognition sites within the P450 structural fold. The role of 10 of these residues, in the B' helix/BC loop, helices F and G, has been studied by site-directed mutagenesis using as a template the soluble sterol 14 alpha-demethylase of known structure, CYP51 from Mycobacterium tuberculosis (MT) and the human orthologue. Single amino acid substitutions of seven residues (Y76, F83, G84, D90, L172, G175, and R194) result in loss of the ability of the mutant MTCYP51 to metabolize lanosterol. Residual activity of D195A is very low, V87A is not expressed as a P450, and A197G has almost 1 order of magnitude increased activity. After purification, all of the mutants show normal spectral properties, heme incorporation, and the ability to be reduced enzymatically and to interact with azole inhibitors. Profound influence on the catalytic activity correlates well with the spectral response to substrate binding, effect of substrate stabilization on the reduced state of the P450, and substrate-enhanced efficiency of enzymatic reduction. Mutagenesis of corresponding residues in human CYP51 implies that the conserved amino acids might be essential for the evolutionary conservation of sterol 14 alpha-demethylation from bacteria to mammals.     

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.     

Differential inhibition of human CYP3A4 and Candida albicans CYP51 with azole antifungal agents

The inhibition by azole antifungals of human cytochrome CYP3A4, the major form of drug metabolising enzyme within the liver, was compared with their inhibitory activity against their target enzyme, Candida albicans sterol 14alpha-demethylase (CYP51), following heterologous expression in Saccharomyces cerevisiae. IC(50) values for ketoconazole and itraconazole CYP3A4 inhibition were 0.25 and 0. 2 microM. These values compared with much lower doses required for the complete inhibition of C. albicans CYP51, where IC(50) values of 0.008 and 0.0076 microM were observed for ketoconazole and itraconazole, respectively. Additionally, stereoselective inhibition of CYP3A4 and CYP51 was observed with enantiomers of the azole antifungal compounds diclobutrazol and SCH39304. In both instances, the RR(+) configuration at their asymmetric carbon centres was most active. Interestingly, the SS(-) enantiomeric form of SCH39304 was inactive and failed to bind CYP3A4, as demonstrable by Type II binding spectra.