Dissertation
THE ROLE OF ALDEHYDE OXIDASE: UNDERSTANDING ITS MECHANISM AND KINETICS
Doctor of Philosophy (PhD), Washington State University
01/2018
Handle:
https://hdl.handle.net/2376/111534
Abstract
Aldehyde oxidase (AOX) is a cytosolic enzyme that belongs to the molybdenum hydroxylase family. AOX remains understudied and is often overlooked as a drug-metabolizing enzyme, which resulted in failure of multiple clinical trials. This research described our efforts to understand the role of AOX in drug metabolism. In the first study, we described the development of an economical and practical microbial-based system expressing human Aldehyde oxidase, addressing the significant variability in existing in vitro systems used in AOX preclinical studies. ecoAO is a cell paste of Moco-producing E. coli strain TP1017, which can be utilized in various applications for AOX research including metabolite screening, biosynthesis, and enzyme kinetics. Moreover, ecoAO system is a new tool to study the role of AOX in drug metabolism. In the second study, we looked into the AOX-catalyzed oxidative and reductive transformations using 5-nitroquinoline (5NQ) as a substrate. 5NQ can undergo both oxidation and reduction reactions, which enabled us to explore both the oxidation and the reductive half reactions at the same time. The results highlighted the presence of two distinct catalytic sites in the enzyme responsible for the 5NQ transformations: a site for oxidation and a separate site for reduction. Finally, we determined the reductive site that was either close to the flavin or to one of the iron-sulfur clusters. Initially, we used diphenyleneiodonium (DPI) to inhibit the reaction occurring near the flavin. However, DPI was a mixed mode inhibitor of human AOX1, and not a selective inhibitor of electron transfer from the flavin. DPI inhibition at the flavin was found to be highly dependent on the oxidative substrate used. Using the appropriate oxidative substrate and 5NQ, we were able to show that the reduction reaction occurs at the flavin site. Moreover, it was supported by the significant decrease observed in the KM of the 5NQ reduction at anaerobic condition.
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Details
- Title
- THE ROLE OF ALDEHYDE OXIDASE: UNDERSTANDING ITS MECHANISM AND KINETICS
- Creators
- Erickson Mesina Paragas
- Contributors
- Jeffrey P Jones (Advisor)Ming Xian (Committee Member)Gregory Crouch (Committee Member)Hector Aguilar-Carreno (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Chemistry, Department of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 136
- Identifiers
- 99900581622601842
- Language
- English
- Resource Type
- Dissertation