念珠藻 Anabaena sp. CH1 核苷二磷酸醣聚磷酸酶的特性探討

Characterization of NDP-sugar Pyrophosphorylases in Anabaena sp. CH1

黃柏融(Po-Jung Huang)

指導教授 ： 張晃猷

國立清華大學/生命科學院/分子醫學研究所/碩士(2012年)

https://doi.org/10.6843/NTHU.2012.00134

摘要

核苷二磷酸醣聚磷酸酶透過催化磷酸醣產生核苷二磷酸醣，生成醣鍵結物。在原核生物中，念珠藻 (Anabaena sp.) 擁有最多核苷二磷酸醣聚磷酸酶的種類，適合作為研究各類核苷二磷酸醣聚磷酸酶在單一物種中的功能與調節性質的模式生物。念珠藻的核苷二磷酸醣聚磷酸酶表達在大腸桿菌中，純化重組蛋白與分析其酵素活性。透過親緣分析顯示為鳥苷二磷酸甘露糖聚磷酸酶的酵素 alr2361CH1 與 alr3400CH1，分別利用鳥苷二磷酸葡萄醣與尿嘧啶二磷酸葡萄糖。而這也是第一次在原核生物中所發現的鳥苷二磷酸葡萄醣聚磷酸酶。all3274CH1 與 alr3400CH1 皆會利用尿嘧啶雙磷酸葡萄糖，其中前者具有較佳的聚磷酸酶活性。有趣的是，all3274CH1 也具有將尿嘧啶雙磷半乳糖轉化為尿嘧啶三磷酸與半乳糖 1-磷酸的能力。特性方面，這些核苷二磷酸醣聚磷酸酶需要鎂離子達到最高活性；除了 alr3400CH1 外，大部分核苷二磷酸醣聚磷酸酶的最佳催化溫度為 47 度。此外，丙酮酸可以作為 alr2361CH1 的異位調節者，進以提昇 alr2361CH1 鳥苷二磷酸葡萄醣聚磷酸酶的活性。總結來說，透過鑑定念珠藻 Anabaena sp. 中的各種核苷二磷酸醣聚磷酸酶的功能特性，可以提供我們細菌生理以及揭示未知生物機能的重要資訊。

關鍵字

念珠藻 ； 核苷二磷酸醣 ； 聚磷酸酶 ； 鳥苷二磷酸葡萄醣

並列摘要

Diverse types of NDP-sugar pyrophosphorylase (NDP-sugar PPase) catalyze phosphosugars to form NDP-sugars, which serve as substrates for glycoconjugates synthesis. Among all prokaryotes analyzed, Anabaena contains the largest number of NDP-sugar PPase types, and can be a good model to understand the interaction and regulation of NDP-sugar PPases in a single organism. All NDP-sugar PPase genes from Anabaena were overexpressed in E. coli, the recombinant proteins were purified, and their enzymatic activities were determined. Enzyme alr2361CH1 and alr3400CH1, both were annotated as a GDP-Man PPase by phylogenetic analysis, were found to utilize GDP-Glc and UDP-Glc, respectively, as the preferred substrate. This finding is remarkable because this is the novel prokaryotic GDP-Glc PPase ever discovered. Both all3274CH1 and alr3400CH1 utilize UDP-Glc as the substrate, although the former exhibited a much higher activity. Interestingly, all3274CH1 is also capable of catalyzing an unusual activity - converting UDP-Gal into UTP and Gal 1-P independent of UDP-Glc. All NDP-sugar PPases in Anabaena requires magnesium ion for their maximum activities, and the optimal temperature for most NDP-PPase were at 47 °C except alr3400CH1. Moreover, pyruvate can serve as an allosteric effector and enhance the GDP-Glc PPase activity of alr2361CH1. To sum up, the characterization of the enzymatic properties of NDP-sugar PPases in Anabaena can provide critical information on the bacterial physiology and reveal previously unknown biological processes.

並列關鍵字

Anabaena sp. ； NDP-sugar ； pyrophosphorylase ； GDP-glucose

參考文獻

1. Ohtsubo, K. and J.D. Marth, Glycosylation in cellular mechanisms of health and disease. Cell, 2006. 126(5): p. 855-67.

2. Weijers, C.A., M.C. Franssen, and G.M. Visser, Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides. Biotechnol Adv, 2008. 26(5): p. 436-56.

3. Szymanski, C.M. and B.W. Wren, Protein glycosylation in bacterial mucosal pathogens. Nat Rev Microbiol, 2005. 3(3): p. 225-37.

4. Lindquist, L., et al., Purification, characterization and HPLC assay of Salmonella glucose-1-phosphate thymidylyl-transferase from the cloned rfbA gene. Eur J Biochem, 1993. 211(3): p. 763-70.

5. Lindqvist, L., et al., Purification, characterization, and high performance liquid chromatography assay of Salmonella glucose-1-phosphate cytidylyltransferase from the cloned rfbF gene. J Biol Chem, 1994. 269(1): p. 122-6.