Structural Biology of Cytochrome P450 Monooxygenases in Arabidopsis Thaliana Phenylpropanoid Pathway
Rupasinghe, Sanjeewa G.
This item is only available for download by members of the University of Illinois community. Students, faculty, and staff at the U of I may log in with your NetID and password to view the item. If you are trying to access an Illinois-restricted dissertation or thesis, you can request a copy through your library's Inter-Library Loan office or purchase a copy directly from ProQuest.
Permalink
https://hdl.handle.net/2142/72483
Description
Title
Structural Biology of Cytochrome P450 Monooxygenases in Arabidopsis Thaliana Phenylpropanoid Pathway
Author(s)
Rupasinghe, Sanjeewa G.
Issue Date
2008
Doctoral Committee Chair(s)
Clayton, David F.
Department of Study
Cell and Developmental Biology
Discipline
Cell and Developmental Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Biophysics, General
Biology, Plant Physiology
Abstract
Plant genomes are rich in cytochrome P450 monooxygenase sequences with 246 full-length genes found in the Arabidopsis thaliana genome. Unlike other enzymes that catalyze substrate modifications, P450s catalyze the activation of oxygen, which then reacts with the nearest substrate atom. Among the many plant P450s characterized to date, four (CYP73A5, CYP75B1, CYP84A1, CYP98A3) catalyze rate-limiting hydroxylation steps in the phenylpropanoid pathway. Homology modeling studies of the P450-substrate interactions in these four P450s have indicated that substrates bind in similar orientations and locations in all four P450s. De novo models of several bacterial and mammalian P450s, built using new alignment strategies and hybrid templates have shown that they resemble their respective crystal structures better than models developed using conventional sequence alignment strategies. With the success of these modeling predictions, virtual screening methods were developed to accelerate the process of defining functionalities for orphan P450s. In these the three-dimensional structures of compounds in metabolic pathways were docked in a number of P450 models and ranked according to the predicted protein-substrate interactions. Site-directed mutagenesis and comparative metabolism studies with CYP98A3 using its natural and alternative substrates have supported predictions on their binding modes. These experiments have clarified the structural specificity of CYP98A3 for p-coumaroyl shikimate and indicated a possible substrate access channel. Procedures have also been developed for the expression of N-terminally truncated forms of CYP98A3 and CYP73A5 suitable for x-ray crystallography. Both proteins have been used in crystallographic trials and are being processed for full-scale structural determinations. Protein expression strategies have also been developed for expression of 13C,15N-labeled full-length microsomal P450s in minimal isotopically enriched media suitable for solid state NMR analysis. CYP98A3 labeled using these methods has generated NMR spectra indicative of a uniformly precipitated protein retaining its secondary structural elements. In addition to pioneering strategies for structure determinations on plant P450s, this work represents the most thorough structural analysis of any plant P450. As plant P450s are numerous and have highly divergent functions, information provided by structural biology studies will have a greater and long lasting effect on P450 biology and its applications.
Use this login method if you
don't
have an
@illinois.edu
email address.
(Oops, I do have one)
IDEALS migrated to a new platform on June 23, 2022. If you created
your account prior to this date, you will have to reset your password
using the forgot-password link below.
