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Date of Award

Spring 2023

Document Type

Masters Thesis

Department or Program Affiliation


Degree Name

Master of Science (MS)



First Advisor

Spiegel, P. Clint

Second Advisor

McCarty, Jay

Third Advisor

Antos, John M.


Cytosine Deaminases (CD) are a class of enzymes found in prokaryotes and fungi that have been studied in the past due to their ability to deaminate the prodrug 5-fluorocytosine (5-FC) producing 5-fluorouracil (5-FU). 5-FU is a common anti-cancer drug that can inhibit DNA synthesis leading to cancer cell death. 5-fluorocytosine can interact with digestive bacteria leading to unwanted side effects for cancer patients. Isocytosine Deaminases (ICD) are enzymes that are of interest in the treatment of cancers using Gene Directed Enzyme Prodrug Therapy (GDEPT). ICDs can deaminate the prodrug 5-fluoroisocytosine (5-FIC) also producing the drug 5-FU.  5-FIC will likely not be recognized by digestive bacteria due to ICDs being less common than CDs resulting in reduced side effects for patients. In this study, the crystal structure of URA4, an isocytosine deaminase, was determined using X-ray crystallography. Using the crystal structure of URA4, ten point mutations were made in the active site to better understand the activity of URA4. Live cell assays have been used to determine the viability of human cancers cells after URA4 and the prodrug 5-FIC were added. The enzyme kinetic characteristics were determined for the mutants using both an absorbance assay and isothermal titration calorimetry assay.




Isocytosine Deaminase, Cancer, X-ray Crystallography, Isothermal Titration Calorimetry


Western Washington University

OCLC Number


Subject – LCSH

Cytosine; X-ray crystallography; Calorimetry; Cancer




masters theses




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