ANALYSIS NOVEL DRUG TARGET ENZYMES IN MYCOBACTERIUM TUBERCULOSIS

UoM administered thesis: Phd

  • Authors:
  • Duyet Le

Abstract

CYP126 and CYP141 are novel cytochrome P450 (CYP, P450) proteins from the humanpathogen Mycobacterium tuberculosis (Mtb). In this thesis, both proteins were successfullyexpressed and characterized spectroscopically, kinetically, and structurally. UV-visiblespectroscopy, 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 CObinding. In contrast, CYP141 has distinct properties, including a mixture of high-spin andlow-spin ferric heme iron states and a Soret band shift to 440 nm on binding CO, instead ofthe typical P450 shift to 450 nm. Reduction potential analysis showed that CYP126 has aquite negative potential (-332 mV vs. NHE), comparable to certain other substrate-free Mtband bacterial P450s, whereas CYP141 has an extremely positive potential (-50 mV) that isapproximately 300 mV higher than those of other Mtb P450s. Both enzymes bind to arange of imidazole and triazole antifungal drugs, inducing a type II (red) spectral shift ofthe ferric heme iron. CYP126 and CYP141 were successfully crystallized, as was the Mtbferredoxin 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. Thestructure of the CYP126-ketoconazole complex was also determined by X-raycrystallography, revealing a monomeric P450 in the crystal and with the ketoconazoleimidazole nitrogen ligated directly to the heme iron, replacing a water molecule found asthe heme iron distal ligand in the resting form of CYP126. To reconstitute an electrontransport system supporting CYP141 and CYP126, endogenous (FprA and FdRflavoprotein dehydrogenases, Fdx1 and Fdx2 ferredoxins) and exogenous (E. coli FLDRflavoprotein dehydrogenase and FLD flavodoxin) redox partner proteins were successfullycloned, expressed and purified, and reconstituted with CYP126, CYP141, and othercharacterized Mtb P450s. These redox partner proteins successfully mediated electrontransport from NAD(P)H to Mtb P450s, with exogenous redox partners typically revealinghigher electron transfer rates than Mtb redox partner proteins. Compound screening forCYP126 allowed identification of several inhibitors and potential substrates from a libraryof 20,000 organic molecules. Approximately 30 compounds were identified based on theirinducing good type I or type II binding spectra. CYP126 interactions with one of the toptype I hits (compound 32027) and the top type II hit (compound 35125) were analyzedbiochemically and biophysically. UV-visible and EPR spectroscopy showed that compound32027 binding to CYP126 induced accumulation of high-spin ferric heme iron, consistentwith 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 withcompounds 32027 and 35125 were successfully crystallized, leading to structural analysis.

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Original languageEnglish
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Award date31 Dec 2011