Purification and crystallization of two 6-phosphoglucose isomerase mutants from Bacillus subtilis

• Main Authors: Chia-Lung Chiang, 江佳龍
• Other Authors: 李天雄
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/19102578521267568306

碩士 === 國立中興大學 === 生物化學研究所 === 95 === Cytosolic 6-phosphoisomerase (PGI, E.C.5.3.1.9) 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.