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    CHARACTERISATION OF CYTOCHROME P450 AZOLE DRUG-RESISTANT STEROL DEMETHYLASE CYP51B1 AND EXPRESSION OF CYP123 AND CYP136 FROM MYCOBACTERIUM TUBERCULOSIS

    Fernandez, Christine

    [Thesis]. Manchester, UK: The University of Manchester; 2011.

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    Abstract

    Tuberculosis (TB) affects nearly a third of the world’s population and has been termed a ‘Global Emergency’ by the WHO. The emergence of multi/extensively drug resistant (M/XDR) strains of Mycobacterium tuberculosis (Mtb), the causative agent of TB, and the increasing incidences of azole drug resistant sterol demethylases (CYP51) from pathogenic fungi has propelled studies to understand mechanisms of azole drug resistance on the drug target CYP51. Since Mtb is devoid of a sterol biosynthetic pathway, the presence and study of CYP51B1 and 19 other Cytochrome P450s in its genome is important to clarify host-pathogen mechanism of infection and the potential of using azole drugs to treat TB. In this study, CYP51B1 from Mtb was used as the model enzyme to study CYP51 mutants from Candida albicans fluconazole-resistant clinical strains. By protein engineering methods, F89H, L100F, S348F, G388S and R391K CYP51B1 mutants were made and azole drug binding properties were investigated using stopped-flow kinetics and static equilibrium methods. Dissociation constant (Kd) values were derived for a range of commercially available azole drugs by fitting the equilibrium binding data to a hyperbolic equation. Kd values for stopped-flow kinetics were derived by plotting observed binding rates (kobs) across different azole drug concentrations against time, followed by fitting multiple kobs data to a linear equation to derive azole drug de-binding (koff) and binding (kon) rate constants – the Kd was obtained by koff/kon. Extinction coefficient for heme b content in mutants and Wild Type (WT) CYP51B1 were an average of ɛ419 = 96.1 mM-1 cm-1. Biochemical characterisation of the mutants were carried out using established experiments on CYP51 – reduction of Fe(III)-heme to Fe(II)-heme, NO binding to Fe(III)-heme, rates of CO-Fe(II) adduct formation and rates of collapse of the P450 to P420 species in the presence of CO and estriol with redox partners from Mtb. In order to elucidate the effects of the above mutations on the iron-heme catalytic region, electron paramagnetic resonance (EPR) experiments were carried out with and without azole drugs. Circular dichroism (CD), differential scanning calorimetry (DSC) and multi-angled laser light scattering (MALLS) analysis confirmed that F89H, R391K and L100F mutants were stable and homogeneous. Crystallogenesis was successful for the above mentioned mutants and atomic structures were obtained for all mutants and WT CYP51B1 (in ligand-bound and substrate-free forms), except for S348F and G388S mutants which were expressed as inclusion bodies and 60% holoenzyme, respectively. Reconstituted catalytic assays to determine the sterol demethylating propensity of the mutants were carried out using redox partners from Mtb or E. coli, and with lanosterol and dihydrolanosterol as the surrogate substrates. Redox potentiometry showed similar potentials to WT for all mutants except for the G388S mutant which was relatively positive (–102 mV). Redox cycling experiments followed by EPR analysis for mutants and WT resulted in a novel P450 high-spin species at g value 5.84 (80 %) which gradually collapsed to the initial low spin state over 48 h. Expression trials were concurrently carried out on two other Mtb P450 genes – CYP123 (Rv0744c) and CYP136 (Rv3059) products of which may have similar functions to CYP51B1 or may share similar redox partners. CYP123 is located on the same operon as CYP51B1 while CYP136 has a 29% sequence identity to another CYP51 from a marine slime bacterium. Although further work is necessary, in this study CYP123 was expressed totally as inclusion bodies while CYP136 was expressed as soluble apoprotein fused with trigger factor chaperone.

    Layman's Abstract

    Tuberculosis (TB) is an airborne infectious disease caused by Mycobacterium tuberculosis (Mtb) which has been called a ‘Global Emergency’ by the World Health Organisation because of increasing numbers of multi-drug resistant (MDR) or extreme multi-drug resistant (XDR) cases. Mtb is a bacterium which contains a relatively large number of proteins from the P450 superfamily which are innately red in colour due to the presence of a catalytically important heme centre anchored within the proteins. One of the 20 P450s in Mtb is called a sterol demethylase (CYP51), an enzyme which is necessary to produce cholesterol in humans, phytosterol in plants and ergosterol in fungi. Amazingly, CYP51B1 does not have a known function in Mtb, since this bacterium does not produce sterols, and yet its molecular structure has been solved. CYP51 however, is the main target for azole-antifungal drugs in patients with fungal infections (candidiasis or aspergillosis caused by Candida albicans and Aspergillus fumigatus, respectively). Concurrently with TB, drug resistant cases in patients with candidiasis are on the rise and researchers have identified important locations at the surface and near the heme in the CYP51 drug target from resistant C. albicans that have changed or mutated. These mutated points have been mapped out onto CYP51B1 of Mtb, which is the only soluble version of this enzyme and therefore can be easily studied. These azole drug resistant mutants have been designated F89H and L100F at the surface (which are also flexible regions), S348F buried in the enzyme, and G388S and R391K near the heme centre. By using various methods, these mutants have been compared to its original non-mutated enzyme (WT) and studied in terms of their biochemical behaviour and binding to azole drugs and a substrate-mimic (estriol). The azole drugs and substrate-mimic interactions with these mutants have been recorded using two methods, one being time-dependent (stopped-flow kinetics) and the other time-independent (equilibrium binding). The values for drug/substrate binding from these two methods were very different from one another. The affinities from stopped-flow values were substantially greater (i.e. weaker binding) than from equilibrium binding values. These values were compared across the different drugs and substrate-mimic to understand the extent of their interactions with the mutants. The heme quantity for each mutant was also measured and the G388S mutant was shown to gradually lose its heme and to exist in two forms, with heme and without heme. This heme loss event substantially influenced G388S mutant biochemical behaviour and interactions with the drugs and substrate-mimic. Mutants at the surface flexible regions and R391K near the heme centre were successfully crystallised in order to solve their molecular structures. These high quality crystals, though small, produced good molecular structures which proved crucial toward understanding the drug interactions near the heme centre and how the azole drug resistant mutations affected these interactions. At the same time, preliminary studies were done for two more P450 proteins from Mtb, which may have similar roles to CYP51B1. They are CYP123 and CYP136, whose functions are also unknown. CYP123 was highly insoluble while CYP136 was partially soluble with heme incorporation problems. Results from this study will eventually help researchers define the function of CYP51B1 in Mtb, explain the mechanism of azole drug resistance in C. albicans and identify potential drugs to address antifungal and TB MDR and XDR problems.

    Bibliographic metadata

    Type of resource:
    Content type:
    Form of thesis:
    Type of submission:
    Degree type:
    Doctor of Philosophy
    Degree programme:
    PhD Biotechnology
    Publication date:
    Location:
    Manchester, UK
    Total pages:
    262
    Abstract:
    Tuberculosis (TB) affects nearly a third of the world’s population and has been termed a ‘Global Emergency’ by the WHO. The emergence of multi/extensively drug resistant (M/XDR) strains of Mycobacterium tuberculosis (Mtb), the causative agent of TB, and the increasing incidences of azole drug resistant sterol demethylases (CYP51) from pathogenic fungi has propelled studies to understand mechanisms of azole drug resistance on the drug target CYP51. Since Mtb is devoid of a sterol biosynthetic pathway, the presence and study of CYP51B1 and 19 other Cytochrome P450s in its genome is important to clarify host-pathogen mechanism of infection and the potential of using azole drugs to treat TB. In this study, CYP51B1 from Mtb was used as the model enzyme to study CYP51 mutants from Candida albicans fluconazole-resistant clinical strains. By protein engineering methods, F89H, L100F, S348F, G388S and R391K CYP51B1 mutants were made and azole drug binding properties were investigated using stopped-flow kinetics and static equilibrium methods. Dissociation constant (Kd) values were derived for a range of commercially available azole drugs by fitting the equilibrium binding data to a hyperbolic equation. Kd values for stopped-flow kinetics were derived by plotting observed binding rates (kobs) across different azole drug concentrations against time, followed by fitting multiple kobs data to a linear equation to derive azole drug de-binding (koff) and binding (kon) rate constants – the Kd was obtained by koff/kon. Extinction coefficient for heme b content in mutants and Wild Type (WT) CYP51B1 were an average of ɛ419 = 96.1 mM-1 cm-1. Biochemical characterisation of the mutants were carried out using established experiments on CYP51 – reduction of Fe(III)-heme to Fe(II)-heme, NO binding to Fe(III)-heme, rates of CO-Fe(II) adduct formation and rates of collapse of the P450 to P420 species in the presence of CO and estriol with redox partners from Mtb. In order to elucidate the effects of the above mutations on the iron-heme catalytic region, electron paramagnetic resonance (EPR) experiments were carried out with and without azole drugs. Circular dichroism (CD), differential scanning calorimetry (DSC) and multi-angled laser light scattering (MALLS) analysis confirmed that F89H, R391K and L100F mutants were stable and homogeneous. Crystallogenesis was successful for the above mentioned mutants and atomic structures were obtained for all mutants and WT CYP51B1 (in ligand-bound and substrate-free forms), except for S348F and G388S mutants which were expressed as inclusion bodies and 60% holoenzyme, respectively. Reconstituted catalytic assays to determine the sterol demethylating propensity of the mutants were carried out using redox partners from Mtb or E. coli, and with lanosterol and dihydrolanosterol as the surrogate substrates. Redox potentiometry showed similar potentials to WT for all mutants except for the G388S mutant which was relatively positive (–102 mV). Redox cycling experiments followed by EPR analysis for mutants and WT resulted in a novel P450 high-spin species at g value 5.84 (80 %) which gradually collapsed to the initial low spin state over 48 h. Expression trials were concurrently carried out on two other Mtb P450 genes – CYP123 (Rv0744c) and CYP136 (Rv3059) products of which may have similar functions to CYP51B1 or may share similar redox partners. CYP123 is located on the same operon as CYP51B1 while CYP136 has a 29% sequence identity to another CYP51 from a marine slime bacterium. Although further work is necessary, in this study CYP123 was expressed totally as inclusion bodies while CYP136 was expressed as soluble apoprotein fused with trigger factor chaperone.
    Layman's abstract:
    Tuberculosis (TB) is an airborne infectious disease caused by Mycobacterium tuberculosis (Mtb) which has been called a ‘Global Emergency’ by the World Health Organisation because of increasing numbers of multi-drug resistant (MDR) or extreme multi-drug resistant (XDR) cases. Mtb is a bacterium which contains a relatively large number of proteins from the P450 superfamily which are innately red in colour due to the presence of a catalytically important heme centre anchored within the proteins. One of the 20 P450s in Mtb is called a sterol demethylase (CYP51), an enzyme which is necessary to produce cholesterol in humans, phytosterol in plants and ergosterol in fungi. Amazingly, CYP51B1 does not have a known function in Mtb, since this bacterium does not produce sterols, and yet its molecular structure has been solved. CYP51 however, is the main target for azole-antifungal drugs in patients with fungal infections (candidiasis or aspergillosis caused by Candida albicans and Aspergillus fumigatus, respectively). Concurrently with TB, drug resistant cases in patients with candidiasis are on the rise and researchers have identified important locations at the surface and near the heme in the CYP51 drug target from resistant C. albicans that have changed or mutated. These mutated points have been mapped out onto CYP51B1 of Mtb, which is the only soluble version of this enzyme and therefore can be easily studied. These azole drug resistant mutants have been designated F89H and L100F at the surface (which are also flexible regions), S348F buried in the enzyme, and G388S and R391K near the heme centre. By using various methods, these mutants have been compared to its original non-mutated enzyme (WT) and studied in terms of their biochemical behaviour and binding to azole drugs and a substrate-mimic (estriol). The azole drugs and substrate-mimic interactions with these mutants have been recorded using two methods, one being time-dependent (stopped-flow kinetics) and the other time-independent (equilibrium binding). The values for drug/substrate binding from these two methods were very different from one another. The affinities from stopped-flow values were substantially greater (i.e. weaker binding) than from equilibrium binding values. These values were compared across the different drugs and substrate-mimic to understand the extent of their interactions with the mutants. The heme quantity for each mutant was also measured and the G388S mutant was shown to gradually lose its heme and to exist in two forms, with heme and without heme. This heme loss event substantially influenced G388S mutant biochemical behaviour and interactions with the drugs and substrate-mimic. Mutants at the surface flexible regions and R391K near the heme centre were successfully crystallised in order to solve their molecular structures. These high quality crystals, though small, produced good molecular structures which proved crucial toward understanding the drug interactions near the heme centre and how the azole drug resistant mutations affected these interactions. At the same time, preliminary studies were done for two more P450 proteins from Mtb, which may have similar roles to CYP51B1. They are CYP123 and CYP136, whose functions are also unknown. CYP123 was highly insoluble while CYP136 was partially soluble with heme incorporation problems. Results from this study will eventually help researchers define the function of CYP51B1 in Mtb, explain the mechanism of azole drug resistance in C. albicans and identify potential drugs to address antifungal and TB MDR and XDR problems.
    Thesis main supervisor(s):
    Thesis advisor(s):
    Language:
    en

    Institutional metadata

    University researcher(s):
    Academic department(s):

    Record metadata

    Manchester eScholar ID:
    uk-ac-man-scw:132661
    Created by:
    Fernandez, Christine
    Created:
    6th October, 2011, 23:25:10
    Last modified by:
    Fernandez, Christine
    Last modified:
    6th March, 2019, 11:31:34

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