- 學位論文
鬆脆桿菌鳥苷二磷酸岩藻糖合成酶之結構分析
Structural analysis of Bacteroides fragilis GDP-fucose synthetase
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摘要
岩藻糖 (fucose) 常修飾在寡醣鏈末端，在許多生理作用上扮演不可或缺的重要性。鬆脆桿菌 (Bacteroides fragilis) 為人類腸道常見的共生菌之一；鬆脆桿菌會由宿主腸胃道表皮細胞上之末端取得岩藻糖，並利用岩藻糖生合成的補救途徑 (salvage pathway) 來合成GDP-L-fucose，以提高表面路易士抗原的表現量，達到躲避宿主的免疫作用。與其他物種不同，鬆脆桿菌將L-fucokinase (FK) 及GDP-L-fucose pyrophosphorylase (GFPP) 合而為一，而具有雙功能之蛋白 (稱為FKP)。本研究以酵素動力學和蛋白質結構來探討這個雙功能蛋白的結構與功能。根據蛋白質序列比對，我們將全長FKP (FL-FKP)、以及含有單一酵素功能的FK (518-949)、GFPP (1-496) 在大腸桿菌上表現、生產、純化後達到 > 95% 的純度，它們酵素動力學分析指出， FL-FKP所含FK活性與FK518-949比較起來，前者在Km (fucose)上比後者低10倍，在kcat (fucose) 和kcat (ATP) 的測量上，全長較單一活性區域高2倍。以GFPP活性作比較，FL-FKP和GFPP1-496兩者在kcat (fucose-1-P; f1p) 及kcat (GTP) 上，前者均較後者高出兩倍。此外，以分析型超高速離心法分析FL-FKP在緩衝溶液中的四級結構大多為三聚體。在x-ray結晶解析上，為了解決GFPP相位問題，我們將SUMO蛋白接至GFPP蛋白N端上並表現得到GFPP單體，並成功解析出它的結構 (解析度2.35 Å)，最後藉由GFPP、FK結構解析出FL-FKP 之結構 (解析度2.37 Å)。我們藉由結構、與其他相似蛋白結構比較及定點突變方式，發現FK活性區段催化是以D762為general base，作用在fucose C-1的羥基上；W599、D601及Q761負責與fucose的結合作用；R592、E751、S719及S720則與ATP的結合有關。這些資訊讓我們提出FK可能催化機制。除此之外，我們嘗試將兩個酵素活性之間的連結區域 (序列431-583) 作部分剔除，並無法表現可溶性之蛋白，發現這個區域提供活性與結構穩定的必要性。
並列摘要
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.
參考文獻
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延伸閱讀
- 吳正國（2014）。鬆脆桿菌鳥苷二磷酸岩藻醣合成酶及人類第二型岩藻醣水解酶之性質探討〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2014.10211
- Huang, P. J. (2012). 念珠藻 Anabaena sp. CH1 核苷二磷酸醣聚磷酸酶的特性探討 [master's thesis, National Tsing Hua University]. Airiti Library. https://doi.org/10.6843/NTHU.2012.00134
- 羅鈞（2011）。人類第二型岩藻醣水解酶之生化性質探討及偵測〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2011.10506
- 杜欣宜（2011）。念珠藻麥芽寡糖苷海藻糖合成酶及水解酶之選殖、表現及特性分析〔碩士論文，國立臺灣師範大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0021-1610201315253453
- Chen, C. L. (2017). Structural studies of glyco-engineered IgG/Fc receptor complex [doctoral dissertation, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU201700712