Defense Date

2009

Document Type

Thesis

Degree Name

Master of Science

Department

Medicinal Chemistry

First Advisor

John C. Hackett

Abstract

P450s are heme containing enzymes which affect oxidation of substrates via catalytic intermediates having transient lifetimes. These oxidative catalytic intermediates are formed by a sequential interplay of electrons and protons at the active site of the enzyme bearing molecular dioxygen. The proton transfer to the active site from bulk solvent is coordinated by an “acid-alcohol” pair of active site residues which are conserved in all P450s. Sterol 14α-demethylases (CYP51) are P450 enzymes which catalyze oxidative deformylation of lanosterol in the cholesterol/ergosterol biosynthetic pathway. Both cholesterol and ergosterol are important regulators of membrane fluidity. CYP51 differs from other P450s in that the acid in the acid-alcohol pair in the active site is replaced by a His residue. This enzyme is present in tuberculosis (TB) causing pathogen Mycobacterium tuberculosis (Mtb). This finding was significant for primarily two reasons. The first one being the baffling presence of CYP51 in Mtb, as Mtb is not known to have any endogenous sterol biosynthetic pathways. The second being that CYP51 is a validated drug target in treating fungal infections. Thus given the global resurgence of multidrug resistant strains of Mtb and the deadly coexsistence of Mtb in immunocompromised HIV patients, CYP51 may be an ideal drug target for new generation of antimycobacterial drugs. The Mtb CYP51 enzyme was chosen to study the proton transfer pathways in the active site based on the outcome of explicit solvent molecular dynamics and hybrid quantum mechanics/molecular mechanics calculations performed in our laboratory. Based on these calculations of CYP51 catalysis, Glu173 was implicated to be the proton source. Proton transfer to the active site occurred by a coordinated shuttling via four water molecules, His259 and Thr260. To experimentally verify the roles of Glu173, His259 and Thr260 they were mutated to alanine and biophysically characterized. Ferredoxin, an accessory protein required to shuttle electrons from NADPH to the CYP51 active site for catalysis, was also cloned using ligation independent cloning. We were successfully able to reconstitute the electron transport chain for CYP51. The mutants were found to differentially bind type I and type II enzymes. Based on biophysical characterization, Thr260 can be implicated to have a role in modulating the spin state of the enzyme. The Mtb CYP51 enzyme was chosen to study the proton transfer pathways in the active site based on the outcome of explicit solvent molecular dynamics and hybrid quantum mechanics/molecular mechanics calculations performed in our laboratory. Based on these calculations of CYP51 catalysis, Glu173 was implicated to be the proton source. Proton transfer to the active site occurred by a coordinated shuttling via four water molecules, His259 and Thr260. To experimentally verify the roles of Glu173, His259 and Thr260 they were mutated to alanine and biophysically characterized. Ferredoxin, an accessory protein required to shuttle electrons from NADPH to the CYP51 active site for catalysis, was also cloned using ligation independent cloning. We were successfully able to reconstitute the electron transport chain for CYP51. The mutants were found to differentially bind type I and type II enzymes. Based on biophysical characterization, Thr260 can be implicated to have a role in modulating the spin state of the enzyme.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

August 2009

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