Rachel, Natalie (2011) Active Site Studies of Arogenate Dehydrogenase & the Purification of its Substrate. Masters thesis, Concordia University.
- Accepted Version
Arogenate dehydrogenase (AD) from the cyanobacterium Synechocystsis sp. PCC6803 is one enzyme within the family of TyrA proteins dedicated to the biosynthesis of L-tyrosine (L-Tyr) and catalyzes the NADP+-dependant oxidative decarboxylation of L-arogenate to L-Tyr. To circumvent some of the difficulties in obtaining L-arogenate using current methodology, we developed an improved methodology to synthesize L-arogenate enzymatically using the recently annotated prephenate aminotransferase from Petunia hybrida. L-arogenate was then purified from major contaminants by high performance liquid chromatography. Guided by sequence alignments and the crystal structure of AD, residues were targeted for the first mutagenesis experiments on this enzyme; the variant proteins were characterized by kinetic, biophysical and computational methods. We identified that His112, a highly conserved residue, is the catalytic hydrogen bond acceptor and is critical for enzyme function, whereas electrostatic interactions afforded by Asp171 appear critical for the overall stability of the enzyme. Both Arg213 and Arg217, a highly conserved residue, appear to play a role in enzyme function, which contradicts the conclusion inferred from the crystal structure. His170, was also identified to be an important active site residue, and likely maintains His112 in a catalytically competent conformation via hydrogen bonding. Ser178, His179, T117 and Gly221 were all also found to be important for enzyme function. Even though the exact roles of these residues require further clarification, our studies have provided valuable insight into how arogenate dehydrogenase functions.
|Divisions:||Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry|
|Item Type:||Thesis (Masters)|
|Degree Name:||M. Sc.|
|Date:||1 December 2011|
|Thesis Supervisor(s):||Turnbull, Joanne|
|Deposited By:||NATALIE RACHEL|
|Deposited On:||20 Jun 2012 15:34|
|Last Modified:||01 Dec 2013 06:38|
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