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Title: Analysis novel drug target enzymes in Mycobacterium tuberculosis
Author: Le, Duyet
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2011
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CYP126 and CYP141 are novel cytochrome P450 (CYP, P450) proteins from the human pathogen Mycobacterium tuberculosis (Mtb). In this thesis, both proteins were successfully expressed and characterized spectroscopically, kinetically, and structurally. UV-visible spectroscopy, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) showed that CYP126 is a normal P450 enzyme with the major oxidized heme (Soret) band at 418 nm, shifting to 450 nm (hence P450) after heme iron reduction and CO binding. In contrast, CYP141 has distinct properties, including a mixture of high-spin and low-spin ferric heme iron states and a Soret band shift to 440 nm on binding CO, instead of the typical P450 shift to 450 nm. Reduction potential analysis showed that CYP126 has a quite negative potential (-332 mV vs. NHE), comparable to certain other substrate-free Mt band bacterial P450s, whereas CYP141 has an extremely positive potential (-50 mV) that is approximately 300 mV higher than those of other Mtb P450s. Both enzymes bind to a range of imidazole and triazole antifungal drugs, inducing a type II (red) spectral shift of the ferric heme iron. CYP126 and CYP141 were successfully crystallized, as was the Mtb ferredoxin Fdx2 - the latter expressed and purified as a potential Mtb P450 redox partner. The CYP126 crystal structure was solved, exhibiting a dimer with one monomer in the "open" form (with respect to active site access) and the other in the "closed" form. The structure of the CYP126-ketoconazole complex was also determined by X-ray crystallography, revealing a monomeric P450 in the crystal and with the ketoconazole imidazole nitrogen ligated directly to the heme iron, replacing a water molecule found as the heme iron distal ligand in the resting form of CYP126. To reconstitute an electron transport system supporting CYP141 and CYP126, endogenous (FprA and FdR flavoprotein dehydrogenases, Fdx1 and Fdx2 ferredoxins) and exogenous (E. coli FLDR flavoprotein dehydrogenase and FLD flavodoxin) redox partner proteins were successfully cloned, expressed and purified, and reconstituted with CYP126, CYP141, and other characterized Mtb P450s. These redox partner proteins successfully mediated electron transport from NAD(P)H to Mtb P450s, with exogenous redox partners typically revealing higher electron transfer rates than Mtb redox partner proteins. Compound screening for CYP126 allowed identification of several inhibitors and potential substrates from a library of 20,000 organic molecules. Approximately 30 compounds were identified based on their inducing good type I or type II binding spectra. CYP126 interactions with one of the top type I hits (compound 32027) and the top type II hit (compound 35125) were analyzed biochemically and biophysically. UV-visible and EPR spectroscopy showed that compound 32027 binding to CYP126 induced accumulation of high-spin ferric heme iron, consistent with type I binding, and elevated the heme iron redox potential from -332 mV to -176 mV, accelerating electron transfer from redox partners to CYP126. The CYP126 complexes with compounds 32027 and 35125 were successfully crystallized, leading to structural analysis.
Supervisor: Munro, Andrew Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Mtb - Mycobacterium tuberculosis