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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:606870 |
| Date | January 2011 |
| Creators | Fernandez, Christine Cheryl |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://www.manchester.ac.uk/escholar/uk-ac-man-scw:171502 |
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