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標題: 定點突變枯草桿菌6-磷酸葡萄糖異構酶純化與結晶
Purification and crystallization of two 6-phosphoglucose isomerase mutants from Bacillus subtilis
作者: 江佳龍
Chiang, Chia-Lung
關鍵字: phosphoglucose isomerase mutants;磷酸葡萄糖異構酶;Bacillus subtilis
出版社: 生物化學研究所
引用: 1.Read, J., Pearce, J., Li, X., Muirhead, H., Chirgwin, J. & Davies, C. (2001). The crystal structure of human phosphoglucose isomerase at 1.6 angstrom resolution: implications for catalytic mechanism, cytokine activity and haemolytic anaemia. J. Mol. Biol. 309, 447-463. 2.Liotta, L. A., Mandler, R., Murano, G., Katz, D. A., Grodon, R. K., Chiang, P. K. & Schiffmann, E. (1986). Tumor cell autocrine motility factor.Proc Natl Acad Sci USA. 83, 3302-3306. 3.Watanabe, H., Carmi, P., Hogen, V., Raz, T., Silletti, S., Nabi, I. R. & Raz, A. (1991).Purification of human tumor cell autocrine motility factor and molecular cloing of its recepotor. J. Biol. Chem. 266, 631-643. 4.Watanabe, H., Takehama, K., Date, M., Shinozaki, T.& Raz, A. (1996). Tumor cell autocrine motility factors is the neuroleukin/phosphoglucose isomerase polypeptide. Cancer Res. 56, 2960-2963. 5.Chou, C. C., Sun, Y. J., Meng, M. & Hsiao, C. D. (2000). The crystal structure of phosphoglucose isomerase/autocrine motility factor/neuroleukin complexed with its carbohydrate phosphate inhibitors suggests its substrate/receptor recognition. J. Biol. Chem. 275(30), 23154-23160. 6.Xu, W., Seiter, K., Feldman, E., Ahmed, T. & Chiao, W. (1996). The differentiaion and matueration mediator for human myeloid leukemia cells shares homology with neuleukin or phosphoglucose isomerase. Blood 87, 4502-4506. 7.Mastsumoto, I., Staub, A., Benoist, C. & Mathis, D. (1999). Arthritis provoked by linked T and B cell recognition of glycolytic enzyme. Science 286, 1732-1735. 8.Meng, M., Lin, H. Y., Hsieh, C. J. & Chen, Y. T. (2001). Functions of the conserved anionic amino acid and those interacting with the substrate phosphate group of phosphoglucose isomerase. FEBS Letters 499, 11-14. 9.Schray, K. J., Benkovic, S. J., Benkovic, P. A. & Rose, I. A. (1973). Catalytic reactions of phosphoglucose isomerase with cyclic forms of glucose 6-phosphate and fructose 6-phosphate. J. Biol. Chem. 248(6), 2210-2224. 10.Jeffery, C. J., Bahnson, B. J., Chien, W., Ringe, D. & Petsko, G. A. (2000). Crystal structure of rabbit phosphoglucose isomerase, a glycolytic enzyme that moonlights as neuroleukin, autocrine motility factor, and differentiation mediator. Biochemistry 39, 955-964. 11.Jeffery, C. J., Hardre, R. & Salmon, L. (2001). Crystal structure of rabbit phosphoglucose isomerase complexed with 5-phospho-D-arabinonate identifies the role of Glu357 in catalysis. Biochemistry 40, 1560-1566. 12.Antelmann, H., Bernhardt, J., Schmid, R., Mach, H., Volker, U. & Hecker, M. (1997). First steps from a two-dimensional protein index towards a response-regulation map for Bacillus subtilis. Electrophoresis 18(8), 1451-63. 13.Vinopal, R. T., Hillman, J. D., Schulman, H., Reznikoff, W. S. & Fraenkel, D. G. (1975). New phosphoglucose isomerase mutants of Escherichia coli. J. Bacteriol. 122(3), 1172-1174. 14.Pflugrath, J. W. (1999). The finer things in X-ray diffraction data collection. Acta Cryst. D55, 1718-1725. 15.李彥梁 (2005).枯草桿菌6-磷酸葡萄糖異構酶突變蛋白質結構測定. 國立中興大學生物化學研究所碩士論文 16.蘇致安 (2003). Bacillus subtilis 6-磷酸葡萄糖異構酵素中影響熱穩定之主要胺基酸的探討. 國立中興大學生物科技學研究所碩士論文 17.周家丞 (1997).磷酸葡萄糖異構酶的純化、結晶與初步X光結晶分析. 國立中興大學農業生物科技學研究所碩士論文
6-磷酸葡萄糖異構酵素(phosphoglucose isomerase, 簡稱PGI ; E. C.催化D-葡萄糖-6-磷酸和D-果糖-6-磷酸之間的可逆異構轉變,該酵素於六碳糖代謝(glycolysis),及糖質新生(gluconeogenesis)反應中擔任樞紐角色。除此之外,研究顯示PGI在細胞外具有neuroleukin-能提昇特定胚胎及感觸神經元的存活,及促始B細胞成熟為抗體分泌細胞; autocrine motility factor-涉及癌細胞的移轉及侵襲,以及differentiation和maturation mediator-能使培養的骨髓血癌H-60細胞分化為白血球等三種功能。
實驗室先前已測定枯草桿菌中不耐熱、全長為450胺基酸的PGI晶體結構,持續研究另兩株定點突變PGI-I116V與M370L蛋白質,經由大量表達、硫酸胺純澱、及Q-sepharose、15Q-sepharose樹脂純化,分別得到濃度為19.64 mg/mL的PGI-I116V,和25.17 mg/mL的PGI-M370L。其中PGI-M370L能以蒸氣昇華法,於100 mM Tris-HCl at pH 7.5, 30 % ethylene glycol, 18﹪PEG400, 100 mM MgCl2, 0.01﹪NaN3, 1 mM ß-Me培養晶體。晶體的X-光繞射品質達2.4 Å,晶胞為a=146.092 Å、b=136.770 Å、c=109.072 Å、ß=119.660 ˚,而空間群為C2,由CCP4套件中Refmac軟體計算的difference map清楚顯示定點突變的L370無誤,目前R值為0.188,而Rfree為0.285,仍需定水分子及進行最後的結構微調計算。 PGI-I116V突變株雖然可以得到晶體,但是解析度為2.8 Å,急凍至-120 ˚K的晶體數據顯示帶有冰晶及殘破的繞射點,目前在增進結晶及晶體抗凍實驗條件。

Cytosolic 6-phosphoisomerase (PGI, E.C. catalyzes the reversible isomerization of D-glucose 6-phosphate and D-fructose 6-phosphate. This conversion reaction is essential in glycolysis and gluconeogenesis. Moreover, studies show that PGI outside the cell functions as neuroleukin, autocrine motility factor, and a differentiation and maturation mediator. Neuroleukin promotes the survival of embryonic and sensory nerve cells; autocrine is related to tumor invasion and metasis; differentiation and maturation mediator induces the differentiation of leukemia H-60 cells to mature monocytes.
Previously we have solved the crystal structure of a wild type thermolabile PGI of 450 a.a. in length from Bacillus subtillus. We extended the study further of two PGI mutants, PGI-I116V and PGI-M370L, which exhibited 4-fold decrease of enzymatic activity than the wild type PGI. These two PGI mutants were expressed in large quantity, precipitated in ammonium sulfate, and purified by Q- and Resource-15Q sepharose chromatography to homogeneity. The final protein concentration of I116V and M370L were 19.64 mg/mL and 25.17 mg/mL respectively. Prismatic crystals of M370L were grown in solution of 100 mM Tris-HCl at pH 7.5, 30﹪ethylene glycol, 18﹪PEG400, 100 mM MgCl2, 0.01﹪NaN3, and 1.0 mM ß-Me by vapor sublimation method. The crystal diffracted to 2.4 Å with unit cell dimensions of a=146.092 Å, b=136.770 Å, c=109.072 Å, ß=119.660 ˚. The space group was C2 with 4 molecules per asymmetric unit. The difference map calculated from the wild type PGI using REFMAC in CCP4 package clearly showed the contour of the mutated Leucine370. The Rwork is 0.188 and the Rfree is 0.285 for the current model without waters added and further refinements were required. Though the crystals of PGI-I116V were grown under the similar condition, the diffraction data extended only to 2.8 Å with high mosaicity and ice rings. Further refinement of the crystallization and anti-freezing conditions were underway.
其他識別: U0005-0908200714513600
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