- Title Information
- Title
- Electrochemical, biochemical, structural studies of cytochrome P450 monooxygenases involved in metabolism of vitamin D
- Name:
Personal
- Name Part
- Rhieu, Steve
- Role
- Role Term:
Text
- creator
- Origin Information
- Copyright Date
- 2011
- Physical Description
- Extent
- xxi, 117 p.
- digitalOrigin
- born digital
- Note
- Thesis (Ph.D. -- Brown University (2011)
- Name:
Personal
- Name Part
- Palmore, G. Tayhas
- Role
- Role Term:
Text
- Director
- Name:
Personal
- Name Part
- Reddy, G. Satyanarayana
- Role
- Role Term:
Text
- Reader
- Name:
Personal
- Name Part
- Pacifici, Domenico
- Role
- Role Term:
Text
- Reader
- Name:
Personal
- Name Part
- Brodsky, Alexander
- Role
- Role Term:
Text
- Reader
- Name:
Personal
- Name Part
- MacDonald, John
- Role
- Role Term:
Text
- Reader
- Name:
Corporate
- Name Part
- Brown University. Biomedical Engineering
- Role
- Role Term:
Text
- sponsor
- Genre (aat)
- theses
- Abstract
- This dissertation examines two cytochrome P450 monooxygenases, namely CYP27B1 and CYP24A1, for their potential applications in biosensors and drug metabolism, respectively. First, the feasibility of using CYP27B1 as a biocatalyst was explored in an attempt to develop an electrochemical biosensor for measuring vitamin D levels. Electrochemical properties of CYP27B1 were characterized using an edge-plane graphite electrode functionalized with synthetic surfactants to facilitate heterogeneous electron transfer. Cyclic voltammetry in a deoxygenated solution revealed excellent redox reversibility with a midpoint potential of -180 � 5 mV (vs. Ag/AgCl) and a rate constant of 3.5 � 0.6 s-1. However, no product was observed by electrode-driven catalysis despite the excellent electron transfer between CYP27B1 and the electrode. Both spectroscopic and biochemical analyses revealed the structural integrity of CYP27B1 is perturbed by the surfactants, thereby inducing a biologically inactive P420 isomer. These results contribute to the understanding of the apparently anomalous behavior of CYPs in bioelectronic devices. Second, the active form of vitamin D3, 1a,25-dihydroxyvitamin D3 (1) displays non-calcemic actions such as antiproliferative activities against certain types of cancer cells. However, the clinical use of 1 was limited by its side effect of hypercalcemia. To alleviate the calcemic side effects of 1, numerous vitamin D analogs were synthesized with an aim to block metabolic inactivation of 1 by CYP24A1 so that their therapeutic effects in target cells can be prolonged. To provide definitive evidence for the role of structural modifications incorporated into the analogs in their metabolic stability against CYP24A1, the metabolism of two synthetic analogs with 16-ene-23-yne modifications was examined using rat CYP24A1 in a reconstituted system. The metabolism study was accompanied by an in silico CYP24A1 crystal structure-calibrated docking analysis to gain an insight into the structural determinants of substrate recognition as a means to understand the metabolic profiles of given analogs. In addition, the metabolism of a less calcemic natural metabolite of 1, namely 1a,25-dihydroxy-3-epi-vitamin D3 (2), was examined. The end product of 2, 3-epi-calcitroic acid, was isolated and identified for the first time.
- Subject
- Topic
- Cytochrome P450
- Subject
- Topic
- Electrode-driven catalysis
- Subject
- Topic
- Vitamin D metabolism
- Subject (FAST)
(authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/1168206")
- Topic
- Vitamin D
- Subject (FAST)
(authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/832013")
- Topic
- Bioelectrochemistry
- Subject (FAST)
(authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/1168207")
- Topic
- Vitamin D--Metabolism
- Record Information
- Record Content Source (marcorg)
- RPB
- Record Creation Date
(encoding="iso8601")
- 20111003
- Language
- Language Term:
Code (ISO639-2B)
- eng
- Language Term:
Text
- English
- Identifier:
DOI
- 10.7301/Z0W957FV
- Access Condition:
rights statement
(href="http://rightsstatements.org/vocab/InC/1.0/")
- In Copyright
- Access Condition:
restriction on access
- Collection is open for research.
- Type of Resource (primo)
- dissertations