碩士 / 國立臺灣大學 / 生化科學研究所 / 104 / Fucosylation is one of the most common modifications of oligosaccharides on glycoproteins and glycolipids. These fucosylated glycans usually function as special epitopes that contribute to specific physiological effects, including host-microbe interactions. Bacteroides fragilis is known to be one of the most common commensal bacteria in the human gastrointestinal tract. To avoid the host’s immune system, B. fragilis uses salvage pathways to utilize fucose from the host to synthesize fucosylated Lewis antigens, which these bacteria then express on the cell surface. In contrast to other species, B. fragilis combines L-fucokinase and GDP-L-fucose pyrophosphorylase into one bifunctional enzyme (called FKP). In this thesis, we studied the function and structure of FKP by enzyme kinetics and x-ray crystallography. The fragments corresponding to the FK domain (518-949 a.a.), the GFPP domain (1-430 a.a.) and the full-length FKP (FL-FKP) were successfully expressed in the E. coli expression system and isolated with greater than 95% purity. The resulting activity assay indicated that Km (fucose) from FL-FKP was ten-fold lower than that from FK584-949 and that kcat (fucose) and kcat (ATP) from FL-FKP were two-fold higher than that from FK. The GFPP activity results showed that kcat (fucose-1-P; f1p) and kcat (GTP) from FL-FKP were 2-fold higher than that from GFPP1-496. According to analytical ultracentrifugation (AUC) analysis, the quaternary structure of FKP was mostly trimeric. Using x-ray crystallography, we solved the phase problem of the structure of GFPP by fusing a SUMO protein to the N terminus of GFPP and then elucidated its structure (resolution 2.35 Å). Finally, we determined the structure of FL-FKP (resolution 2.37 Å) using the GFPP and FK domain structures. To ascertain the catalytic mechanisms of FK, we used the structure, superimposed structure and site-directed mutagenesis, and proposed that D762 acts as a general base for interacting with the fucose C-1 OH group, W599, D601, and Q761 are responsible for binding to fucose, and R592, E751, S719, and S720 are involved with binding to ATP. Moreover, protein expressions of the partially truncated linker of FKP (431-583 a.a.) are found to result in inclusion bodies, showing that this region is essential for structural intrgrity or/and stability.
| Identifer | oai:union.ndltd.org:TW/104NTU05103105 |
| Date | January 2016 |
| Creators | Hung-Yu Chiang, 江宏宇 |
| Contributors | Chun-Hung Lin, 林俊宏 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 75 |
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