Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23795
標題: 測定枯草桿菌6-磷酸葡萄糖異構酶的突變體晶體結構
Structure determination of the phosphoglucose isomerase mutant from Bacillus subtilis
作者: 李彥樑
Lee, Yan-Liang
關鍵字: 6-磷酸葡萄糖異構酶;phosphoglucose isomerase;葡萄糖-6-磷酸;果糖-6-磷酸;枯草桿菌;活性半衰期;結構;熱穩定性;氣相擴散法;不對稱單元;單位晶格;突變株;PGI;glusocse-6-phosphate;G6P;fructose-6-phosphate;F6P;Bacillus subtilis;PgiBS;PgiB;PgiA;catalytic half-life;structure;thermo stability;X-ray;hanging drop vapor diffusion method;Self-rotation;Matthew's coefficient;CNS;SA omit map;|Fo|-|Fc|;asymmetric unit;unit cell;mutant
出版社: 生物化學研究所
摘要: 
6-磷酸葡萄糖異構酶 (phosphoglucose isomerase, 簡稱PGI;E.C. 5.3.1.9) 催化葡萄糖-6-磷酸與果糖-6-磷酸兩受質異構物間的可逆異構轉換,該反應作用於糖解作用與糖質新生作用中,並能再生五碳糖循環所需代謝起始物。PGI作用於葡萄糖代謝途徑的樞紐位置,為調節葡萄糖能量利用的重要酵素。本計劃研究的枯草桿菌PgiBS,全長為451個胺基酸,與已知結構的PgiB (PDB code:2PGI) 序列相似性為83 %,然而PgiBS熱穩定性不如PgiB。於62 ℃下,PgiBS的活性半衰期約為0.5分鐘,而PgiB的半衰期長達87分鐘。計劃欲解析PgiBS蛋白質晶體結構,再由PgiBS與PgiB的結構差異探討蛋白質熱穩定性關聯。已大量表達PgiBS突變株蛋白質並純化至純度>95 %。PgiBS突變株晶體於包含有15 % PEG400與30 % ethylene glycol的100 mM Tris (pH 6.5) 結晶溶液中,利用氣相擴散法培養。PgiBS突變株晶體使用Cu Kα X-ray光源 (波長1.5418 Å) 收集解析度超過1.9 Å的完整數據。PgiBS突變株晶體的空間群為 C2,單位晶格尺寸a=145.72 Å、b=135.96 Å、c=109.10 Å及β=119.42°。Self-rotation函數分析推測分子排列具有222點群對稱,而Matthew’s coefficient 2.3 Å3/D顯示每個不對稱單元有4個分子,晶體溶液含量為46.8 %。結構測定使用分子取代法,以PgiB已知結構為搜尋模板,使用CCP4套件中的MOREP計算。計算得到的最佳解為每個不對稱單元有4個分子、相關係數54.9與R-factor 44.3 %。分子模型建構經由數次CNS結構幾何最佳化與O程式建構循環,並以SA omit電子密度圖和|Fo|-|Fc|電子密度圖驗證模型幾何與序列一致性。PgiBS突變株最終分子模型,其R-factor為19.1 %而Rfree為23.26 % (10 % test set)。比對PgiBS突變株與PgiB (PDB code:2PGI) 分子結構,顯示兩者整體上十分近似 (主鏈Cα r.m.s. 0.61 Å),但位於PgiBS突變株催化區內的PgiBS序列204-216與307-313兩區段,與結合受質態的PgiB (PDB code:1C7R) 較為相似。經由PgiBS突變株結構的成功測定,提供探討蛋白質熱穩定性與結構關聯的良好機會。

Phosphoglucose isomerase (PGI; E.C. 5.3.1.9) catalyzes the reversible isomerization of D-glusocse-6-phosphate (G6P) and D-fructose-6-phosphate (F6P) in glycolysis and gluconeogenesis pathways, and in the regeneration of starting material in the pentose phosphate pathway. PGI plays an important role in utilizing glucose as energy source at the pivot point of these glucose-related metabolism pathways. PGI (PgiBS) from Bacillus subtilis is 451 a.a. in length with 83 % sequence homology to PgiB of known crystal structure (PDB code:2PGI). PgiBS, however, is a thermal-labile protein. The catalytic half-life of PgiBS is about 0.5 minutes at 62 ℃, much less than 87 minutes of PgiB. We aimed to determine the structure of PgiBS, and to study the thermal stability of PgiBS and PgiB. The PgiBS mutant was overexpressed and purified to > 95 % homogeneity by chromatography methodology. The crystals of PgiBS mutant were grown by hanging drop vapor diffusion method in 100 mM Tris (pH 6.5) crystallization solution containing 15 % PEG400 and 30 % ethylene glycol. A complete diffraction data was collected beyond 1.9 Å resolution using monochromized Cu Kα X-ray (1.5418 Å). The crystal belongs to spacegroup C2 with unit cell dimensions a=145.72 Å, b=135.96 Å, c=109.10 Å, and β=119.42°. Self-rotation function analysis data showed crystal packing of 222 point group symmetry, and the Matthew's coefficient of 2.3 Å3/D indicated 4 molecules per asymmetric unit and water content of 46.8%. The structure was determined by molecular replacement method using known the PgiB structure as search model. The MOREP in CCP4 package found a best solution of four molecules with the correlation coefficient of 54.9 and an R-factor of 44.3%. The model was built through cycles of map-fitting using program O and structure refinement using CNS package and REFMAC. The geometry and PgiBS mutant sequence identity of the model were checked through series of SA omit maps and|Fo|-|Fc|maps. The final PgiBS mutant model has an R-factor of 19.1 % and Rfree of 23.26 % (10 % test set). The structures of PgiBS mutant and PgiB (PDB code:2PGI) show high similarity in overall fold (Cα r.m.s. 0.61 Å). Two regions of PgiBS mutant model in the catalytic site, PgiBS mutant sequence 204-216 and 307-313, are similar to the structure of ligand-binding PgiB (PDB code:1C7R). The PgiBS mutant structure provides an opportunity to find the possible relation between the structure and thermo-lability.
URI: http://hdl.handle.net/11455/23795
Appears in Collections:生物化學研究所

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