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Title: Evidence that cytochrome b{sub 5} acts as a redox donor in CYP17A1 mediated androgen synthesis

Abstract

Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second C−C lyase step, at the expense of glucocorticoid production. Cytochrome b{sub 5} (cyt b{sub 5}) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b{sub 5} increases androgen biosynthesis. Cyt b{sub 5} could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b{sub 5} could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b{sub 5}, we generated a redox-inactive form of cyt b{sub 5}, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b{sub 5}), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stablemore » membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b{sub 5} were enhanced ∼5 fold as compared to reactions in the absence of cyt b{sub 5}. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b{sub 5}. Upon addition of Mn-b{sub 5} to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b{sub 5}-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal Fe−S vibrational frequency. Thus, although Mn-b{sub 5} binds to CYP17A1, it is unable to enhance the lyase reaction, strongly suggesting that cyt b{sub 5} has a redox effector role in enhancement of the CYP17A1 mediated lyase reaction necessary for androgen synthesis. - Highlights: • Cyt b{sub 5} role in human CYP17A1 mediated androgen synthesis was probed in Nanodiscs. • Native cyt b{sub 5} enhances androgen synthesis by CYP17A1. • Redox inactive Mn cyt b{sub 5} does not enhance androgen synthesis by CYP17A1. • Interactions with Cyt b{sub 5} perturb Fe−S and heme Raman modes of CYP17A1. • Cyt b{sub 5} acts as a redox donor for CYP17A1 mediated androgen synthesis.« less

Authors:
 [1];  [2]; ;  [1];  [2];  [1];  [3]
  1. Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL (United States)
  2. Department of Chemistry, Marquette University, Milwaukee, WI (United States)
  3. (United States)
Publication Date:
OSTI Identifier:
22606157
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 477; Journal Issue: 2; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AFFINITY; ANCHORS; BIOSYNTHESIS; CASTRATION; DRUGS; GLUCOCORTICOIDS; HEME; HYDROXYANDROSTENONE; HYDROXYLATION; HYDROXYPREGNENONE; IN VIVO; IRON; LYASES; MANGANESE; NEOPLASMS; PROSTATE; STOICHIOMETRY; SUBSTRATES

Citation Formats

Duggal, Ruchia, Liu, Yilin, Gregory, Michael C., Denisov, Ilia G., Kincaid, James R., Sligar, Stephen G., E-mail: s-sligar@illinois.edu, and Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL. Evidence that cytochrome b{sub 5} acts as a redox donor in CYP17A1 mediated androgen synthesis. United States: N. p., 2016. Web. doi:10.1016/J.BBRC.2016.06.043.
Duggal, Ruchia, Liu, Yilin, Gregory, Michael C., Denisov, Ilia G., Kincaid, James R., Sligar, Stephen G., E-mail: s-sligar@illinois.edu, & Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL. Evidence that cytochrome b{sub 5} acts as a redox donor in CYP17A1 mediated androgen synthesis. United States. doi:10.1016/J.BBRC.2016.06.043.
Duggal, Ruchia, Liu, Yilin, Gregory, Michael C., Denisov, Ilia G., Kincaid, James R., Sligar, Stephen G., E-mail: s-sligar@illinois.edu, and Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL. 2016. "Evidence that cytochrome b{sub 5} acts as a redox donor in CYP17A1 mediated androgen synthesis". United States. doi:10.1016/J.BBRC.2016.06.043.
@article{osti_22606157,
title = {Evidence that cytochrome b{sub 5} acts as a redox donor in CYP17A1 mediated androgen synthesis},
author = {Duggal, Ruchia and Liu, Yilin and Gregory, Michael C. and Denisov, Ilia G. and Kincaid, James R. and Sligar, Stephen G., E-mail: s-sligar@illinois.edu and Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL},
abstractNote = {Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second C−C lyase step, at the expense of glucocorticoid production. Cytochrome b{sub 5} (cyt b{sub 5}) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b{sub 5} increases androgen biosynthesis. Cyt b{sub 5} could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b{sub 5} could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b{sub 5}, we generated a redox-inactive form of cyt b{sub 5}, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b{sub 5}), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b{sub 5} were enhanced ∼5 fold as compared to reactions in the absence of cyt b{sub 5}. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b{sub 5}. Upon addition of Mn-b{sub 5} to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b{sub 5}-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal Fe−S vibrational frequency. Thus, although Mn-b{sub 5} binds to CYP17A1, it is unable to enhance the lyase reaction, strongly suggesting that cyt b{sub 5} has a redox effector role in enhancement of the CYP17A1 mediated lyase reaction necessary for androgen synthesis. - Highlights: • Cyt b{sub 5} role in human CYP17A1 mediated androgen synthesis was probed in Nanodiscs. • Native cyt b{sub 5} enhances androgen synthesis by CYP17A1. • Redox inactive Mn cyt b{sub 5} does not enhance androgen synthesis by CYP17A1. • Interactions with Cyt b{sub 5} perturb Fe−S and heme Raman modes of CYP17A1. • Cyt b{sub 5} acts as a redox donor for CYP17A1 mediated androgen synthesis.},
doi = {10.1016/J.BBRC.2016.06.043},
journal = {Biochemical and Biophysical Research Communications},
number = 2,
volume = 477,
place = {United States},
year = 2016,
month = 8
}
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