Structural and Functional Investigations of Cytochrome P450 Enzymes from Mycobacterium Species
Date
2022
Authors
Mohamed, Hebatalla Ahmed Ibrahim
Editors
Advisors
Bell, Stephen
Bruning, John
Bruning, John
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Abstract
Cytochrome P450s (CYP) are heme-monooxygenase enzymes, responsible for the catalytic hydroxylation of a large variety of organic molecules. Mycobacterium marinum a bacterial pathogen of fish and frogs, has a larger genome than the closely related pathogenic bacterium, Mycobacterium tuberculosis, and Mycobacterium ulcerans, (the causative agents of human tuberculosis and Buruli ulcer, respectively). The genome of M. marinum contains 47 CYP encoding genes. Mycobacteria have a member of the sterol demethylase CYP51 family of cytochrome P450 enzymes which have a key role in the metabolic synthesis of steroids across most of the kingdoms of life. In the course of this work, two CYP51 enzymes from different Mycobacteria were produced and studied. The steroid metabolising (CYP51 family members) enzyme from Mycobacterium marinum was compared to its counterpart from Mycobacterium tuberculosis. Steroid substrates were found to bind differently to the enzymes. The catalytic activity of both CYP51 enzymes with lanosterol and related steroids was studied. Both were able to oxidatively demethylate lanosterol to generate 14-demethylanosterol. The addition of smaller steroids and other ligands to both enzymes also displayed different responses. Structural characterisation of CYP51 from Mycobacterium marinum was undertaken by X-ray crystallography and determined to a resolution of 1.98 Å. Structural changes were observed compared to the equivalent M. tuberculosis enzyme; these changes could be correlated to the different steroid binding properties. Mycobacterial CYP51 enzymes demonstrated important differences to the eukaryotic CYP51 enzymes and the bacterial CYP51 enzyme of Methylococcus capsulatus. CYP105Q4 from M. marinum was characterised in order to better define its function. The structural homolog search and the phylogenetic analysis of the CYP105Q4 amino acid sequence indicates it closely resembles other structurally characterised CYP105 enzymes. CYP105Q4 bound several nitrogen-donor ligand inhibitor-like molecules. In some instances, the inhibitors bound form of the enzyme showed complex behaviour. None of the hydrophobic substrates we tested were able to displace the sixth axial water ligand in the active site, however, oleic acid and amphotericin B induced a small blue shift of the UV-vis spectrum of the enzyme. CYP105Q4 was structurally characterised by X-ray crystallography and showed conformational changes in the position of the helices, and the loop connecting them compared to other CYP105 enzymes. The enzyme had an open conformation and a disordered B/C loop suggesting flexibility in this region which is responsible for substrate recognition. CYP147G1 from M. marinum which can selectively oxidise linear fatty acids was produced and purified in improved yield in an attempt to expand our understanding of this enzyme. Compounds which were anticipated to bind to CYP51 enzymes, were tested with CYP147G1 and induced a type I shift. Also, various nitrogen-donor molecules were able to bind to CYP147G1 resulting in a red-shift of the main band in the UV-vis absorbance spectrum (Soret band). Ammonium sulfate precipitation was used to fractionate the protein prior to purification by chromatography procedures followed by size-exclusion chromatography to further purify CYP147G1. A number of commercially available sparse matrix crystal screens were employed in an attempt to crystallise CYP147G1, identifying four separate conditions that yielded crystals. Further optimisation screens were also set up. Of these screens CYP147G1 crystals were obtained and were sent to the synchrotron for X-ray diffraction analysis. However, none of the crystals diffracted in a sufficient quality for further study. There is a great interest in designing molecules to block/inhibit the function of cytochrome P450 enzymes as a method for disease treatment, therefore investigate the binding of ligands to P450 enzymes and a full understanding to the spectral responses are required. The interactions of nitrogen-containing ligands with the ferric and ferrous forms of the enzyme are important as reduction of the heme and prevention of oxygen binding to the ferrous form would be two of the steps they aim to inhibit. The binding of nitrogen-containing ligands to the ferric and ferrous forms of a selection of cytochrome P450 enzyme was investigated using UV-vis absorbance spectroscopy. Differences in UV-vis spectrum and therefore the heme environment were observed in the ligand bound ferrous forms of various P450 enzyme/ligand combinations. A ferrous species associated with intense and sharp α-band and a Soret band at ~427 nm was detected. None of the P450 spectral experiments showed a single species of 6-coordinate ferrous thiolate form (with a Soret band at ~442-447 nm). It was also observed that the Fe-N ligand bond could break on reduction forming a 5-coordinate high-spin ferrous species (411- 414 nm). In some P450 enzymes, the ferrous form appeared to be oxidised back to the ferric form on addition of the ligand. Formation of mixtures of these species was also observed across the enzymes we investigated. The type of buffer (Tris versus phosphate) could also result in spectral differences in the ferrous form of some of the P450 enzymes. This study provides new information into the spectra and species that can be obtained when studying ligand binding to heme-thiolate enzymes. We also hope it inspires more spectroscopic investigations with more advanced techniques into the identity of these species and a better understanding of the spectroscopic responses observed. The generation of a structure-based pharmacophore model was hypothesised for the CYP51 family 14⍺-sterol demethylase P450 enzymes followed by virtual screening in hope to identify new bacterial CYP51 inhibitors. Docking studies were employed to predict the correct binding poses of inhibitors inside the active site of CYP51 enzymes. We identified the most promising CYP51 inhibitors for further investigation. These could be employed to design ligands with enhanced inhibitory potencies against CYP51 and related cytochrome P450 enzymes.
School/Discipline
School of Physics, Chemistry and Earth Sciences
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Physics, Chemistry and Earth Sciences, 2023
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