Degree Type

Dissertation

Date of Award

2015

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Major

Molecular, Cellular and Developmental Biology

First Advisor

Reuben J. Peters

Abstract

Labdane-related diterpenoids are a large group of natural products (over 7,000 known) with applications in many industries. The cyclization of the general diterpene precursor, (E,E,E)-geranylgeranyl diphosphate, into various bicyclic diterpene precursors catalyzed by class II diterpene cyclases characterizes labdane-related diterpenoid biosynthesis. The catalytic acid in the protonation-initiated cyclization reaction has been previously identified, however the catalytic base was not known. Using ent-copalyl diphosphate synthase from Arabidopsis thaliana, involved in gibberellin phytohormone biosynthesis, site-directed mutagenesis studies have uncovered the catalytic base responsible for deprotonation, which consists of a water molecule ligated, in part, by the side-chains from a highly conserved His and Asn dyad. Substitution of smaller side chains for either of these residues leads to addition of water, resulting in production of the hydroxylated variant of ent-copalyl diphosphate, ent-copal-8-ol diphosphate. Whereas, substitution of aromatic residues for the His blocks water access, and stabilizes the carbocation intermediate, thereby facilitating rearrangement around the initially formed hydrocarbon bicycle, resulting in formation of (-)-kolavenyl diphosphate. These mutants provide insights into class II diterpene cyclase catalysis, represent novel enzymatic activities and illustrate how the plasticity of class II diterpene cyclases underlies the observed diversity of labdane-related diterpenoids, thus, uncovering the BASEs for catalysis.

DOI

https://doi.org/10.31274/etd-180810-4177

Copyright Owner

Kevin Charles Potter

Language

en

File Format

application/pdf

File Size

171 pages

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