Date of Award
2006
Degree Type
Thesis
Degree Name
Master of Science
Program
Biology
Supervisor
Dr. Susanne Kohalmi
Second Advisor
Dr. Mark Bernards
Third Advisor
Dr. Denis Maxwell
Abstract
Six putative arogenate dehydratases (ADT) isoforms have been identified in Arabidopsis thaliana, but are listed online as prephenate dehydratases (PDTs) based on sequence similarity. To determine whether the putative Arabidopsis ADTs recognise arogenate, prephenate, or both, individual isoforms were analysed using a molecular and biochemical approach. An extensive in silico analysis showed that while putative plant ADT sequences form a distinct group from bacterial and yeast sequences, they share all sequence motifs that were identified through mutagenic studies as being essential for function. Putative ADTs were cloned into a Saccharomyces cerevisiae and two Escherichia coli expression systems, and expressed as His-fusion proteins. Putative ADTs were successfully purified and assayed against either arogenate or prephenate to test enzyme specificity, however, results were inconclusive due to apparent non-enzymatic conversion. A second approach tested the function of two putative ADTs by complementation of a PDT knockout in S. cerevisiae. This test showed that ADT1 could complement the PDT of S. cerevisiae while ADT3 could not, suggesting that there may be functional differences between these ADT isoforms.
Recommended Citation
Corea, Oliver Ryan Ardiel, "MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF ARABIDOPSIS AROGENATE DEHYDRATASES" (2006). Digitized Theses. 4980.
https://ir.lib.uwo.ca/digitizedtheses/4980