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Title:Mechanistic investigation of human cytochromes P450 involved in hormone biosynthesis
Author(s):Duggal, Ruchia
Director of Research:Sligar, Stephen G
Doctoral Committee Chair(s):Sligar, Stephen G
Doctoral Committee Member(s):Gennis, Robert B; Tajkhorshid, Emad; Schuler, Mary A
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Cytochrome P450 17A1 (CYP17A1)
Cytochrome b5
CYP19A1
Abstract:CYP17A1 and CYP19A1 are the key cytochromes P450 involved in steroidogenesis. Mutations causing hypo- or hyper-activation of both these enzymes results in diseased states, including cancers. In addition to carrying out mono-oxygenation reactions, both CYP17A1 and CYP19A1 also catalyze carbon-carbon lyase reactions. There is significant interest in developing therapeutics capable of specifically targeting the CYP17A1 lyase reaction to target androgen synthesis without impacting glucocorticoid production, and understanding of CYP19A1 reaction mechanism could similarly help guide design of mechanism based inhibitors for certain breast cancers. To interrogate mechanistic aspects of both these enzymes, I used Nanodisc technology for reconstituting active forms of these proteins in functional complexes with their effector proteins in a native-like membrane environment. Cytochrome P450 17A1 (CYP17A1) is a multi-functional enzyme, catalyzing synthesis of glucocorticoid precursors by hydroxylation of pregnene nucleus, and androgen biosynthesis by a second C-C lyase step, at the expense of glucocorticoid production. It has been recently confirmed in our lab that when 17-hydroxy-pregnenolone is a substrate, CYP17A1 lyase reaction proceeds through a non-conventional hemiketal intermediate. However, it remains unknown if the alternate substrate, 17-hydroxy-progesterone (OH-PROG) is transformed to androstenedione via this novel mechanism, by the traditional compound I (Cpd I) mechanism, or by a combination of the two. To this end, I investigated kinetic solvent isotope effects on steady state turnover for lyase reaction on OH-PROG. Further, cryo-trapped peroxo-anion intermediate was spectroscopically followed as the samples were thermally annealed. Resonance Raman spectroscopy (rR) on the trapped intermediate was also performed. Our results strongly indicate that the CYP17A1 mediated OH-PROG lyase proceeds through a hemiketal intermediate. Cytochrome b5 (cyt b5) is a small heme-protein, known to enhance the rate of the CYP17A1 lyase reaction by ~5 fold, while having little effect on hydroxylation. Investigation with a redox inactive form of cyt b5 indicated that for lyase enhancement, cyt b5 critically requires a redox effector role. In order to investigate the electron transfer by cyt b5 to CYP17A1, I employed stopped flow spectroscopy and compared the CYP17A1 reduction rates by cyt b5 that by cytochrome P450 reductase (CPR). RR was also performed on CYP17A1—cyt b5 complex investigate any conformational perturbations. Taken together, our results suggest that cyt b5 critically requires a redox transfer role is essential to enhance lyase reaction, and it reduces CYP17A1 oxy-complex ~10fold faster than CPR. RR on oxyferrous CYP17A1—cyt b5 complex indicated subtle conformational changes in the active site, which were heavily dependent on the identity of the substrate. CYP19A1 produces estrogens in a three-step reaction, the first two being hydroxylation steps, while the third step comprises a lyase step. The hydroxylation reactions are expected to proceed through the conventional Cpd I intermediate, but the active intermediate for the lyase step is not known. We investigated kinetic solvent isotope effects on the steady state turnover for the three-step reaction, and our results suggest that CYP19A1 lyase chemistry, unlike CYP17A1, proceeds through the Cpd I intermediate. My thesis is a mechanistic study of the androgenic CYP17A1 and the estrogenic CYP19A1. I investigated the active intermediates in the final lyase step by employing various biochemical and biophysical techniques to Nanodisc reconstituted enzymes. While CYP19A1 lyase step is suggested to proceed through the Cpd I intermediate, in the case of CYP17A1 lyase reaction, a hemiketal intermediate is implicated. Furthermore, the biological regulator of androgen synthesis, cyt b5, acts as a faster redox donor than CPR in the lyase reaction cycle, and also exerts subtle conformational changes dependent on the identity of the substrate in the active site.
Issue Date:2017-10-20
Type:Text
URI:http://hdl.handle.net/2142/99474
Rights Information:Copyright 2017 Ruchia Duggal
Date Available in IDEALS:2018-03-13
2020-03-14
Date Deposited:2017-12


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