Graduation Year

2024

Document Type

Campus Only Senior Thesis

Degree Name

Bachelor of Arts

Department

Biochemistry

Reader 1

Bethany Caulkins

Reader 2

Katie Purvis-Roberts

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2023 Quinlin Z Dixon-Lim

Abstract

The enzyme Ornithine Decarboxylase (ODC) catalyzes the formation of the polyamine putrescine, through the decarboxylation of L-ornithine, which leads to the formation of the polyamines spermidine and spermine. Polyamines are essential in the body, functioning as antioxidants, controlling cell proliferation, and stabilizing the structure of DNA, amongst other roles. ODC is pyridoxal 5′-phosphate (PLP)-dependent, meaning it requires PLP to bind in the active site of ODC before it is enzymatically active. PLP is a biochemically common cofactor, making it a particularly interesting molecule to study, because it undergoes many different conformations in a diverse range of reactions. ODC is active as a homodimer, meaning its active site is composed of residues from two identical subunits. The current treatment for Human African trypanosomiasis, or Sleeping Sickness, is α-difluoromethylornithine (DFMO), a known inhibitor of ODC. Prior to the discovery of this treatment, Sleeping Sickness was often fatal. Due to its role in cell proliferation, ODC has been linked to carcinogenesis, and is therefore a promising drug target candidate for chemoprevention. Inhibitors such as DFMO have already shown promise in aiding cancer treatment in partnership with chemotherapy. This underscores the need for a detailed understanding of the enzyme and the way it functions during catalysis. A robust protocol for purifying the Saccharomyces cerevisiae (Brewer’s yeast) form of ODC from BL21 E. coli cells was developed in order to achieve three ultimate goals: analyze data on the kinetics of ODC inhibitors and substrate analogs, elucidate its 3D structure using solid-state NMR, and better understand the exact conformational changes involved in its mechanism. In order to confirm protein purity, multiple Western Blots were attempted, resulting in a protein band roughly half of the molecular weight of an expected ODC monomer. This result contradicted much of the previous evidence that suggested a 50 kDa monomer of ODC, as well as the 100 kDa active dimerized form, were successfully purified. The difficulty in obtaining a pure sample of identified S. cerevisiae ODC perhaps reveals why little literature exists about this enigmatic form.

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