University of Illinois Urbana-Champaign

Understanding the mechanism of androgen biosynthesis

Gregory, Michael Carlton

Content Files
GREGORY-DISSERTATION-2016.pdf

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https://hdl.handle.net/2142/95533

Description

Title
Understanding the mechanism of androgen biosynthesis
Author(s)
Gregory, Michael Carlton
Issue Date
2016-08-01
Director of Research (if dissertation) or Advisor (if thesis)
Sligar, Stephen G.
Doctoral Committee Chair(s)
Sligar, Stephen G.
Committee Member(s)
  • Das, Aditi
  • Gennis, Robert B.
  • Morrissey, James H.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2017-03-01T17:00:47Z
Keyword(s)
  • CYP17A1
  • Nanodisc
  • P450
Abstract
Cytochrome P45017A1 (CYP17A1) is a multifunctional steroidogenic enzyme responsible for the 17-hydroxylation of pregnenolone and progesterone as well as the subsequent 17,20 carbon-carbon bond scission of its hydroxylated products in a reaction that constitutes the first committed step of androgen formation. Though over 40 years have passed since discovery of this enzyme, key questions regarding the nature of the reactive intermediate responsible for androgen formation have heretofore remained unanswered. Specifically, while the hydroxylase activity of CYP17A1 is expected to proceed through standard P450 ferryl-oxene intermediate, identity of the iron-oxygen species involved in carbon-carbon bond scission has until now remained elusive. This dissertation documents the steady state kinetics of CYP17A1 catalysis as well as detailed characterization of its heme environment in the ferric, oxy-ferrous, and peroxo- states by resonance Raman spectroscopy in order to answer this essential question. Presence of an inverse solvent isotope effect during dehydroepiandrosterone formation, as well as functional lyase catalysis by a CYP17A1 mutant possessing an impaired proton-delivery network, strongly support the hypothesis that a ferric peroxoanion is responsible for androgen formation. Additionally, resonance Raman spectroscopy of the oxy-ferrous form of this enzyme revealed that identity of the carbon-3 substituent on the pregnene- nucleus directs the 17-OH group to hydrogen bond with the Fe-O-O ligand in a manner that alternately promotes or impedes efficient lyase catalysis via this nucleophilic pathway. Finally, resonance Raman of the during stepwise thermal annealing of the cryotrapped peroxo- state permitted identification of a new peroxo hemiacetal intermediate species initiated by nuclophilic attack on the substrate molecule by the peroxoanion. As a whole, this work details the structural and functional properties of CYP17A1 active in human androgen biosynthesis.
Graduation Semester
2016-12
Type of Resource
text
Permalink
http://hdl.handle.net/2142/95533
Copyright and License Information
Copyright 2016 Michael C Gregory

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