The Experts below are selected from a list of 57 Experts worldwide ranked by ideXlab platform
Steven L Kelly - One of the best experts on this subject based on the ideXlab platform.
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a novel Sterol 14alpha Demethylase ferredoxin fusion protein mccyp51fx from methylococcus capsulatus represents a new class of the cytochrome p450 superfamily
Journal of Biological Chemistry, 2002Co-Authors: Colin J Jackson, David C Lamb, Diane E Kelly, N J Manning, Timothy H Marczylo, Andrew G S Warrilow, David J Lowe, Steven L KellyAbstract:Sterol 14α-Demethylase encoded by CYP51 is a member of the cytochrome P450 (CYP) superfamily of enzymes and has been shown to have an essential role in Sterol biosynthesis in eukaryotes, with orthologues recently being described in some bacteria. Examination of the genome sequence data for the proteobacterium Methylococcus capsulatus, a bacterial species known to produce Sterol, revealed the presence of a single CYP with strong homology to CYP51, particularly to a form in Mycobacterium tuberculosis. ThisM. capsulatus CYP51 protein represents a new class of CYP consisting of the CYP domain naturally fused to a ferredoxin domain at the C terminus via an alanine-rich linker. Expression of the M. capsulatus MCCYP51FX fusion in Escherichia coliyielded a P450, which, when purified to homogeneity, had the predicted molecular mass ∼62 kDa on SDS/PAGE and bound lanoSterol as a putative substrate. Sterol 14α-Demethylase activity was shown (0.24 nmol of lanoSterol metabolized per minute per nanomole of MCCYP51FX fusion) by gas chromatography/mass spectrometry with the activity dependent upon the presence of ferredoxin reductase and NADPH. Our unique findings describe a new class of naturally existing cytochrome P450, which will provide pivotal information for CYP structure/function in general.
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Sterol 14alpha Demethylase activity in streptomyces coelicolor a3 2 is associated with an unusual member of the cyp51 gene family
Biochemical Journal, 2002Co-Authors: David C Lamb, Larissa M Podust, Michael R Waterman, Diane E Kelly, Kay Fowler, Tobias Kieser, N J Manning, Steven L KellyAbstract:The annotation of the genome sequence of Streptomyces coelicolor A3(2) revealed a cytochrome P450 (CYP) resembling various Sterol 14alpha-Demethylases (CYP51). The putative CYP open reading frame (SC7E4.20) was cloned with a tetrahistidine tag appended to the C-terminus and expressed in Escherichia coli. Protein purified to electrophoretic homogeneity was observed to bind the 14-methylated Sterols lanoSterol and 24-methylene-24,25-dihydrolanoSterol (24-MDL). Reconstitution experiments with E. coli reductase partners confirmed activity in 14alpha-demethylation for 24-MDL, but not lanoSterol. An S. coelicolor A3(2) mutant containing a transposon insertion in the CYP51 gene, which will abolish synthesis of the functional haemoprotein, was isolated as a viable strain, the first time a CYP51 has been identified as non-essential. The role of this CYP in bacteria is intriguing. No Sterol product was detected in non-saponifiable cell extracts of the parent S. coelicolor A3(2) strain or of the mutant. S. coelicolor A3(2) CYP51 contains very few of the conserved CYP51 residues and, even though it can catalyse 14alpha-demethylation, it probably has another function in Streptomyces. We propose that it is a member of a new CYP51 subfamily.
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the g464s amino acid substitution in candida albicans Sterol 14alpha Demethylase causes fluconazole resistance in the clinic through reduced affinity
Biochemical and Biophysical Research Communications, 1999Co-Authors: Steven L Kelly, David C Lamb, Juergen Loeffler, Herman Einsele, Diane E KellyAbstract:Abstract Fluconazole selectively inhibits fungal Sterol 14α-Demethylase, a cytochrome P450 enzyme found in plants, animals, fungi, and Mycobacteria. The mutation G464S, observed in the heme-binding domain of Sterol 14α-Demethylase in clinical strains of fluconazole-resistant Candida albicans, is shown here to cause resistance through substantially reducing the inhibitory effect of fluconazole and is associated with perturbation of the heme environment as indicated by spectral data. The protein exhibits 42% of the maximal enzymatic rate of the wild-type protein allowing continued production of the end product of fungal Sterol biosynthesis, ergoSterol, in resistant strains. This mutation may cause these phenotypes through altering the heme location, thus changing the ability of residues above the heme to bind the drug effectively. This perturbation would also account for the observation of reduced Sterol Demethylase catalytic activity by changing the location of the 14α-methyl group in relation to oxygen-bound heme during the catalytic cycle.
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stereoselective interaction of sch 39304 a triazole with Sterol 14alpha Demethylase of aspergillus fumigatus
Journal of Antimicrobial Chemotherapy, 1997Co-Authors: K Venkateswarlu, Steven L KellyAbstract:The inhibitory activity of SCH 39304 and its enantiomers on radial growth and on the target enzyme, Sterol 14α-Demethylase, in Aspergillus fumigatus was studied to assess the role of stereochemistry in the efficacy of the drug. SCH 39304 and the RR (+) enantiomer were active in inhibiting the growth while no inhibition in the growth was observed with the SS (-) enantiomer. The MIC of SCH 39304 for the growth was about twice that of the RR (+) enantiomer. The differences in IC 50 s of SCH 39304 and its enantiomers for cell-free ergoSterol biosynthesis correlated with their variations in MICs and type II binding spectra indicated the SS (-) enantiomer failed to bind to microsomal P450. These results show that the difference between SS (-) and RR (+) enantiomers in interacting with the target enzyme is the cause for significant difference in the potency between these two forms.
Michael R Waterman - One of the best experts on this subject based on the ideXlab platform.
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Sterol 14alpha Demethylase cyp51 as a therapeutic target for human trypanosomiasis and leishmaniasis
Current Topics in Medicinal Chemistry, 2011Co-Authors: Galina I Lepesheva, Michael R WatermanAbstract:Pathogenic protozoa threaten lives of several hundred million people throughout the world and are responsible for large numbers of deaths globally. The parasites are transmitted to humans by insect vectors, more than a hundred of infected mammalian species forming reservoir. With human migrations, HIV-coinfections, and blood bank contamination the diseases are now spreading beyond the endemic tropical countries, being found in all parts of the world including the USA, Canada and Europe. In spite of the widely appreciated magnitude of this health problem, current treatment for sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi) and leishmaniasis (Leishmania spp.) remains unsatisfactory. The drugs are decades old, their efficacy and safety profiles are unacceptable. This review describes Sterol 14α-Demethylase, an essential enzyme in Sterol biosynthesis in eukaryotes and clinical target for antifungal azoles, as a promising target for antiprotozoan chemotherapy. While several antifungal azoles have been proven active against Trypanosomatidae and are under consideration as antiprotozoan agents, crystal structures of Sterol 14α-Demethylases from three protozoan pathogens, Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum provide the basis for the development of new, highly potent and pathogen-specific drugs with rationally optimized pharmacological properties.
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structural insights into inhibition of Sterol 14α Demethylase in the human pathogen trypanosoma cruzi
Journal of Biological Chemistry, 2010Co-Authors: Galina I Lepesheva, Tatiana Y Hargrove, S Anderson, Yuliya Y Kleshchenko, Vyacheslav Furtak, Zdzislaw Wawrzak, Fernando Villalta, Michael R WatermanAbstract:Trypanosoma cruzi causes Chagas disease (American trypanosomiasis), which threatens the lives of millions of people and remains incurable in its chronic stage. The antifungal drug posaconazole that blocks Sterol biosynthesis in the parasite is the only compound entering clinical trials for the chronic form of this infection. Crystal structures of the drug target enzyme, Trypanosoma cruzi Sterol 14alpha-Demethylase (CYP51), complexed with posaconazole, another antifungal agent fluconazole and an experimental inhibitor, (R)-4'-chloro-N-(1-(2,4-dichlorophenyl)-2-(1H-imid-azol-1-yl)ethyl)biphenyl-4-carboxamide (VNF), allow prediction of important chemical features that enhance the drug potencies. Combined with comparative analysis of inhibitor binding parameters, influence on the catalytic activity of the trypanosomal enzyme and its human counterpart, and their cellular effects at different stages of the Trypanosoma cruzi life cycle, the structural data provide a molecular background to CYP51 inhibition and azole resistance and enlighten the path for directed design of new, more potent and selective drugs to develop an efficient treatment for Chagas disease.
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estriol bound and ligand free structures of Sterol 14alpha Demethylase
Structure, 2004Co-Authors: Larissa M Podust, Liudmila V Yermalitskaya, Galina I Lepesheva, Vladimir N Podust, Enrique A Dalmasso, Michael R WatermanAbstract:Abstract Sterol 14α-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 3β-hydroxy group of a Sterol, and identified F78 as a key discriminator between 4α-methylated and 4α,β-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.
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Sterol 14alpha Demethylase activity in streptomyces coelicolor a3 2 is associated with an unusual member of the cyp51 gene family
Biochemical Journal, 2002Co-Authors: David C Lamb, Larissa M Podust, Michael R Waterman, Diane E Kelly, Kay Fowler, Tobias Kieser, N J Manning, Steven L KellyAbstract:The annotation of the genome sequence of Streptomyces coelicolor A3(2) revealed a cytochrome P450 (CYP) resembling various Sterol 14alpha-Demethylases (CYP51). The putative CYP open reading frame (SC7E4.20) was cloned with a tetrahistidine tag appended to the C-terminus and expressed in Escherichia coli. Protein purified to electrophoretic homogeneity was observed to bind the 14-methylated Sterols lanoSterol and 24-methylene-24,25-dihydrolanoSterol (24-MDL). Reconstitution experiments with E. coli reductase partners confirmed activity in 14alpha-demethylation for 24-MDL, but not lanoSterol. An S. coelicolor A3(2) mutant containing a transposon insertion in the CYP51 gene, which will abolish synthesis of the functional haemoprotein, was isolated as a viable strain, the first time a CYP51 has been identified as non-essential. The role of this CYP in bacteria is intriguing. No Sterol product was detected in non-saponifiable cell extracts of the parent S. coelicolor A3(2) strain or of the mutant. S. coelicolor A3(2) CYP51 contains very few of the conserved CYP51 residues and, even though it can catalyse 14alpha-demethylation, it probably has another function in Streptomyces. We propose that it is a member of a new CYP51 subfamily.
Patrice Le Pape - One of the best experts on this subject based on the ideXlab platform.
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the amino acid substitution n136y in candida albicans Sterol 14alpha Demethylase is involved in fluconazole resistance
Medical Mycology, 2016Co-Authors: Nidia Alvarezrueda, Audrey Fleury, Cedric Loge, Fabrice Pagniez, Estelle Robert, Florent Morio, Patrice Le PapeAbstract:Resistance to fluconazole antifungal is an ongoing impediment to a successful treatment of Candida albicans infections. One of the most prevalent mechanisms leading to azole resistance is genetic alterations of the 14α-Demethylase, the target of azole antifungals, through point mutations. Site-directed mutagenesis and molecular modeling of 14α-Demethylase rationalize biological data about the role of protein substitutions in the azole treatment failure. In this work, we investigated the role of N136Y substitution by site-directed mutagenesis into Pichia pastoris guided by structural analysis. Single amino acid substitutions were created by site-directed mutagenesis into P. pastoris with C. albicans ERG11 gene as template. In vitro susceptibility of P. pastoris transformants expressing wild-type and mutants to azole compounds was determined by CLSI M27-A2 and spot agar methods. The fluconazole effect on ergoSterol biosynthesis was analyzed by gas chromatography-mass spectrometry. By microdilution and spot tests, N136Y transformants showed a reduced in vitro susceptibility to fluconazole compared to wild-type controls. As expected, ergoSterol/lanoSterol ratios were higher in N136Y transformants compared to the wild-type controls after treatment with fluconazole. Molecular modeling suggests that residue Asn136 located within the first mutation hot spot, could play a role during heme and azole binding. These results provide new insights into the structural basis for 14α-Demethylase-azole interaction and could guide the design of novel azole antifungals.
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amino acid substitutions at the major insertion loop of candida albicans Sterol 14alpha Demethylase are involved in fluconazole resistance
PLOS ONE, 2011Co-Authors: Nidia Alvarezrueda, Audrey Fleury, Fabrice Pagniez, Florent Morio, Louis Noel Gastinel, Patrice Le PapeAbstract:Background: In the fungal pathogen Candida albicans, amino acid substitutions of 14alpha-Demethylase (CaErg11p, CaCYP51) are associated with azole antifungals resistance. This is an area of research which is very dynamic, since the stakes concern the screening of new antifungals which circumvent resistance. The impact of amino acid substitutions on azole interaction has been postulated by homology modeling in comparison to the crystal structure of Mycobacterium tuberculosis (MT-CYP51). Modeling of amino acid residues situated between positions 428 to 459 remains difficult to explain to date, because they are in a major insertion loop specifically present in fungal species. Methodology/Principal Finding: Fluconazole resistance of clinical isolates displaying Y447H and V456I novel CaErg11p substitutions confirmed in vivo in a murine model of disseminated candidiasis. Y447H and V456I implication into fluconazole resistance was then studied by site-directed mutagenesis of wild-type CaErg11p and by heterogeneously expression into the Pichia pastoris model. CLSI modified tests showed that V447H and V456I are responsible for an 8-fold increase in fluconazole MICs of P. pastoris mutants compared to the wild-type controls. Moreover, mutants showed a sustained capacity for producing ergoSterol, even in the presence of fluconazole. Based on these biological results, we are the first to propose a hybrid homology structure-function model of Ca-CYP51 using 3 different homology modeling programs. The variable position of the protein insertion loop, using different liganded or non-liganded templates of recently solved CYP51 structures, suggests its inherent flexibility. Mapping of recognized azole-resistant substitutions indicated that the flexibility of this region is probably enhanced by the relatively high glycine content of the consensus. Conclusions/Significance: The results highlight the potential role of the insertion loop in azole resistance in the human pathogen C. albicans. This new data should be taken into consideration for future studies aimed at designing new antifungal agents, which circumvent azole resistance.
Diane E Kelly - One of the best experts on this subject based on the ideXlab platform.
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a novel Sterol 14alpha Demethylase ferredoxin fusion protein mccyp51fx from methylococcus capsulatus represents a new class of the cytochrome p450 superfamily
Journal of Biological Chemistry, 2002Co-Authors: Colin J Jackson, David C Lamb, Diane E Kelly, N J Manning, Timothy H Marczylo, Andrew G S Warrilow, David J Lowe, Steven L KellyAbstract:Sterol 14α-Demethylase encoded by CYP51 is a member of the cytochrome P450 (CYP) superfamily of enzymes and has been shown to have an essential role in Sterol biosynthesis in eukaryotes, with orthologues recently being described in some bacteria. Examination of the genome sequence data for the proteobacterium Methylococcus capsulatus, a bacterial species known to produce Sterol, revealed the presence of a single CYP with strong homology to CYP51, particularly to a form in Mycobacterium tuberculosis. ThisM. capsulatus CYP51 protein represents a new class of CYP consisting of the CYP domain naturally fused to a ferredoxin domain at the C terminus via an alanine-rich linker. Expression of the M. capsulatus MCCYP51FX fusion in Escherichia coliyielded a P450, which, when purified to homogeneity, had the predicted molecular mass ∼62 kDa on SDS/PAGE and bound lanoSterol as a putative substrate. Sterol 14α-Demethylase activity was shown (0.24 nmol of lanoSterol metabolized per minute per nanomole of MCCYP51FX fusion) by gas chromatography/mass spectrometry with the activity dependent upon the presence of ferredoxin reductase and NADPH. Our unique findings describe a new class of naturally existing cytochrome P450, which will provide pivotal information for CYP structure/function in general.
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Sterol 14alpha Demethylase activity in streptomyces coelicolor a3 2 is associated with an unusual member of the cyp51 gene family
Biochemical Journal, 2002Co-Authors: David C Lamb, Larissa M Podust, Michael R Waterman, Diane E Kelly, Kay Fowler, Tobias Kieser, N J Manning, Steven L KellyAbstract:The annotation of the genome sequence of Streptomyces coelicolor A3(2) revealed a cytochrome P450 (CYP) resembling various Sterol 14alpha-Demethylases (CYP51). The putative CYP open reading frame (SC7E4.20) was cloned with a tetrahistidine tag appended to the C-terminus and expressed in Escherichia coli. Protein purified to electrophoretic homogeneity was observed to bind the 14-methylated Sterols lanoSterol and 24-methylene-24,25-dihydrolanoSterol (24-MDL). Reconstitution experiments with E. coli reductase partners confirmed activity in 14alpha-demethylation for 24-MDL, but not lanoSterol. An S. coelicolor A3(2) mutant containing a transposon insertion in the CYP51 gene, which will abolish synthesis of the functional haemoprotein, was isolated as a viable strain, the first time a CYP51 has been identified as non-essential. The role of this CYP in bacteria is intriguing. No Sterol product was detected in non-saponifiable cell extracts of the parent S. coelicolor A3(2) strain or of the mutant. S. coelicolor A3(2) CYP51 contains very few of the conserved CYP51 residues and, even though it can catalyse 14alpha-demethylation, it probably has another function in Streptomyces. We propose that it is a member of a new CYP51 subfamily.
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the g464s amino acid substitution in candida albicans Sterol 14alpha Demethylase causes fluconazole resistance in the clinic through reduced affinity
Biochemical and Biophysical Research Communications, 1999Co-Authors: Steven L Kelly, David C Lamb, Juergen Loeffler, Herman Einsele, Diane E KellyAbstract:Abstract Fluconazole selectively inhibits fungal Sterol 14α-Demethylase, a cytochrome P450 enzyme found in plants, animals, fungi, and Mycobacteria. The mutation G464S, observed in the heme-binding domain of Sterol 14α-Demethylase in clinical strains of fluconazole-resistant Candida albicans, is shown here to cause resistance through substantially reducing the inhibitory effect of fluconazole and is associated with perturbation of the heme environment as indicated by spectral data. The protein exhibits 42% of the maximal enzymatic rate of the wild-type protein allowing continued production of the end product of fungal Sterol biosynthesis, ergoSterol, in resistant strains. This mutation may cause these phenotypes through altering the heme location, thus changing the ability of residues above the heme to bind the drug effectively. This perturbation would also account for the observation of reduced Sterol Demethylase catalytic activity by changing the location of the 14α-methyl group in relation to oxygen-bound heme during the catalytic cycle.
David C Lamb - One of the best experts on this subject based on the ideXlab platform.
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a novel Sterol 14alpha Demethylase ferredoxin fusion protein mccyp51fx from methylococcus capsulatus represents a new class of the cytochrome p450 superfamily
Journal of Biological Chemistry, 2002Co-Authors: Colin J Jackson, David C Lamb, Diane E Kelly, N J Manning, Timothy H Marczylo, Andrew G S Warrilow, David J Lowe, Steven L KellyAbstract:Sterol 14α-Demethylase encoded by CYP51 is a member of the cytochrome P450 (CYP) superfamily of enzymes and has been shown to have an essential role in Sterol biosynthesis in eukaryotes, with orthologues recently being described in some bacteria. Examination of the genome sequence data for the proteobacterium Methylococcus capsulatus, a bacterial species known to produce Sterol, revealed the presence of a single CYP with strong homology to CYP51, particularly to a form in Mycobacterium tuberculosis. ThisM. capsulatus CYP51 protein represents a new class of CYP consisting of the CYP domain naturally fused to a ferredoxin domain at the C terminus via an alanine-rich linker. Expression of the M. capsulatus MCCYP51FX fusion in Escherichia coliyielded a P450, which, when purified to homogeneity, had the predicted molecular mass ∼62 kDa on SDS/PAGE and bound lanoSterol as a putative substrate. Sterol 14α-Demethylase activity was shown (0.24 nmol of lanoSterol metabolized per minute per nanomole of MCCYP51FX fusion) by gas chromatography/mass spectrometry with the activity dependent upon the presence of ferredoxin reductase and NADPH. Our unique findings describe a new class of naturally existing cytochrome P450, which will provide pivotal information for CYP structure/function in general.
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Sterol 14alpha Demethylase activity in streptomyces coelicolor a3 2 is associated with an unusual member of the cyp51 gene family
Biochemical Journal, 2002Co-Authors: David C Lamb, Larissa M Podust, Michael R Waterman, Diane E Kelly, Kay Fowler, Tobias Kieser, N J Manning, Steven L KellyAbstract:The annotation of the genome sequence of Streptomyces coelicolor A3(2) revealed a cytochrome P450 (CYP) resembling various Sterol 14alpha-Demethylases (CYP51). The putative CYP open reading frame (SC7E4.20) was cloned with a tetrahistidine tag appended to the C-terminus and expressed in Escherichia coli. Protein purified to electrophoretic homogeneity was observed to bind the 14-methylated Sterols lanoSterol and 24-methylene-24,25-dihydrolanoSterol (24-MDL). Reconstitution experiments with E. coli reductase partners confirmed activity in 14alpha-demethylation for 24-MDL, but not lanoSterol. An S. coelicolor A3(2) mutant containing a transposon insertion in the CYP51 gene, which will abolish synthesis of the functional haemoprotein, was isolated as a viable strain, the first time a CYP51 has been identified as non-essential. The role of this CYP in bacteria is intriguing. No Sterol product was detected in non-saponifiable cell extracts of the parent S. coelicolor A3(2) strain or of the mutant. S. coelicolor A3(2) CYP51 contains very few of the conserved CYP51 residues and, even though it can catalyse 14alpha-demethylation, it probably has another function in Streptomyces. We propose that it is a member of a new CYP51 subfamily.
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the g464s amino acid substitution in candida albicans Sterol 14alpha Demethylase causes fluconazole resistance in the clinic through reduced affinity
Biochemical and Biophysical Research Communications, 1999Co-Authors: Steven L Kelly, David C Lamb, Juergen Loeffler, Herman Einsele, Diane E KellyAbstract:Abstract Fluconazole selectively inhibits fungal Sterol 14α-Demethylase, a cytochrome P450 enzyme found in plants, animals, fungi, and Mycobacteria. The mutation G464S, observed in the heme-binding domain of Sterol 14α-Demethylase in clinical strains of fluconazole-resistant Candida albicans, is shown here to cause resistance through substantially reducing the inhibitory effect of fluconazole and is associated with perturbation of the heme environment as indicated by spectral data. The protein exhibits 42% of the maximal enzymatic rate of the wild-type protein allowing continued production of the end product of fungal Sterol biosynthesis, ergoSterol, in resistant strains. This mutation may cause these phenotypes through altering the heme location, thus changing the ability of residues above the heme to bind the drug effectively. This perturbation would also account for the observation of reduced Sterol Demethylase catalytic activity by changing the location of the 14α-methyl group in relation to oxygen-bound heme during the catalytic cycle.