Biological Sciences, School of
School of Biological Sciences: Dissertations, Theses, and Student Research
First Advisor
Hideaki Moriyama
Committee Members
Eduardo Romero Camacho, Eric Weaver, Luwen Zhang
Date of this Version
7-2025
Document Type
Thesis
Citation
A thesis presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Master of Science
Major: Biological Sciences
Under the supervision of Professor Hideaki Moriyama
Lincoln, Nebraska, July 2025
Abstract
Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is an enzyme involved in the pyrimidine biosynthesis pathway, a key component of cellular DNA metabolism. It regulates intracellular uracil carrying triphosphate levels by hydrolyzing dUTP, thereby providing a substrate for thymidylate synthase (TS). The effective inhibition of dUTPase is expected to enhance TS-targeted chemotherapy by promoting thymine-less apoptosis.
Although the reaction mechanism of dUTPase has been studied, the human nuclear homotrimeric form remains complex, as all three subunits cooperatively form a single active site. As a result, the detailed molecular dynamics of its catalysis are still not fully understood. In this study, I investigated the effect of substituting arginine with lysine at position 85 in the human dUTPase nuclear isoform; this modification is commonly employed in protein engineering to prevent thrombin cleavage while maintaining enzymatic function. I constructed three-dimensional structural models of the wild-type and mutant (Arg85Lys) enzymes and analyzed their structural characteristics manually. The wild-type enzyme formed five polar contacts at the Arg85 residue, whereas the Arg85Lys mutant’s contacts were more ambiguous. This reduction likely compromises the transient stabilization of the C-terminal region, which acts as a lid over the active site, potentially affecting intermediate transition states during catalysis.
To experimentally validate the effect of the Arg85Lys substitution, I expressed and purified wild-type and mutant dUTPase in Escherichia coli and measured their enzymatic activities using a colorimetric assay. The mutant exhibited approximately two to three times the Km value of the wild-type, indicating increased substrate affinity. Further structural analysis based on the experimental data revealed incomplete ordering of the active site in the mutant, supporting the hypothesis that C-terminal stabilization is essential for catalysis. These findings suggest that the C-terminal region could serve as a novel target for dUTPase inhibition. Interestingly, the Arg-to-Lys substitution investigated in this study also occurs naturally in lentiviruses infecting livestock, implying that it may represent an evolutionarily conserved mechanism for modulating enzyme activity
Advisor: Hideaki Moriyama
Comments
Copyright 2025, Aaron DeLay. Used by permission