Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB) and poses a global threat to human health. A third of the world’s population is infected with Mtb. Multi-drug resistant and extensively drug resistant Mtb strains are widespread and development of new drugs is urgently needed to treat drug resistant TB. This thesis focuses on the Mtb cytochrome P450 (P450) enzymes CYP126A1 and CYP143A1. P450s are heme-binding enzymes that catalyse activation of molecular oxygen and the oxidation of substrates bound close to the heme. CYP126A1 and CYP143A1 are “orphans” in terms of their functional characterization, but potential drug targets in view of ability of azole-based P450 inhibitors to inhibit growth and viability of Mtb. The CYP126A1 and CYP143A1 genes were cloned and expressed in Escherichia coli. Expression conditions and strains were optimised to maximise soluble protein production and methods were developed to purify the P450s using affinity, ion exchange and size exclusion chromatography. Both P450s were shown to bind heme b, and heme was shown to be axially coordinated by a cysteine thiolate and a water molecule in both cases using UV-visible and electron paramagnetic resonance (EPR) spectroscopy. Both P450s bound carbon monoxide (CO) in their reduced forms to produce heme Fe2+-CO complexes with absorption maxima at ~450 nm – characteristic of P450s. CYP126A1 and CYP143A1 bound avidly to a range of inhibitors, including several azole drugs. As examples, binding constant (Kd) values of 13.8 µM and 21.9 µM were determined for clotrimazole and econazole with CYP143A1; while ketoconazole bound CYP126A1 with a Kd of 0.20 µM. Each of these drugs is very effective in inhibiting Mtb growth. EPR confirmed inhibitory coordination of both P450s by azole drug nitrogen atoms; though indirect coordination via a retained axial water ligand may also occur in some cases. Extinction coefficients were determined as έ420 = 125 mM-1 cm-1 (CYP126A1) and έ415 = 105 mM-1 cm-1 (CYP143A1). CYP126A1’s heme iron redox potential was shown to be unusually positive (E°’ = -80 mV). Light scattering studies showed CYP126A1 to be a monodisperse, monomeric protein. CYP143A1 is also mainly a monomer, but with a small proportion of an oligomeric form. Despite its polydispersity, CYP143A1 was crystallized and its structure solved by X-ray diffraction to a resolution of 1.9 Å, using molecular replacement with the Mtb P450 CYP142A1. A limited compound screen of typical P450 substrates failed to provide “hits” to identify CYP143A1 substrate selectivity, but the presence of polyethylene glycol in the CYP143A1 active site in crystals suggests fatty acids as potential substrates. CYP126A1 was crystallized for studies to identify binding modes of small molecules (“fragments”) identified to interact with CYP126A1 by NMR. Crystal structures of CYP126A1 in complex with two such fragments (NMR401 and NMR343) were determined to ~2.0 Å resolution in ongoing research to build Mtb P450 isoform-specific inhibitors. Compounds identified as CYP126A1 substrates/inhibitors identified by high-throughput screening were validated by UV-visible titrations with the P450, and binding modes and affinity established. In conclusion, this thesis provides novel insights into the biochemical, biophysical and structural properties of two novel Mtb P450s that are potential targets for new anti-TB drugs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:634939 |
| Date | January 2015 |
| Creators | Swami, Shalini |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/structure-and-biochemistry-of-the-orphan-cytochrome-p450s-cyp126a1-and-cyp143a1-from-the-human-pathogen-mycobacterium-tuberculosis(a8dc4eea-678c-419a-a3b3-206253c400b7).html |
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