Date of Graduation
5-2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Chemistry (PhD)
Degree Level
Graduate
Department
Chemistry & Biochemistry
Advisor/Mentor
Sakon, Joshua
Committee Member
Thallapuranam, Suresh
Second Committee Member
Millet, Francis S.
Third Committee Member
Archer, Crystal
Fourth Committee Member
Kim, Jin-Woo
Keywords
Collagen; Collagenase; Mechanism; NMR; SAXS; X-ray crystallography
Abstract
In biological systems, Hathewaya (Clostridium) histolytica secretes collagenases that degrade structural collagen in host tissues and cause tissue necrosis. Despite their detrimental effects, these enzymes have significant therapeutic potential by effectively digesting various collagen subtypes to treat connective tissue disorders. The bacterium produces two homologous collagenases, ColG and ColH, with a catalytic module crucial for hydrolyzing collagen and binding domains, including collagen-binding domains (CBDs) and polycystic kidney disease-like domains (PKDs), for substrate interaction.
Although the crystal structure of the catalytic module of collagenase G (ColG) has been determined, shedding light on its chew-and-digest mechanism, the structure of collagenase H (ColH) remains unknown, limiting a deeper understanding of its catalytic mechanism. In this study, the structure of the ColH catalytic module was resolved in an unprecedented “closed” conformation using X-ray crystallography. Heteronuclear Single Quantum Coherence (HSQC) Nuclear Magnetic Resonance (NMR) spectroscopy further elucidated how ColH recognizes and unwinds collagen triple helix for endopeptidase activity. Small-angle X-ray scattering (SAXS) demonstrated how ColH binds to mini-collagen to feed the catalytic module with the substrate for digestion. Together, these findings provide a comprehensive understanding of ColH’s role in collagen degradation, which would help develop more effective therapeutic agents.
Also, a heparin-crosslinked membrane platform was developed for the large-scale purification of viral vectors. This platform can capture adeno-associated virus serotype 2 (AAV2) particles and release them on demand, which could be helpful for gene therapy applications.
Citation
Bonsu, A. O. (2025). Elucidating the Mechanisms of Collagenase H from Hathewaya (Clostridium) histolytica in Collagen Degradation and Developing a Cross-linked Platform for Efficient Capture and Release of Adeno-Associated Virus Serotype 2 (AAV2). Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/5644
