球形红细菌无机焦磷酸酶的原核表达、纯化及初步分析

无机焦磷酸酶(inorganic pyrophosphatase,PPase)水解在许多生物大分子的生物合成过程中产生焦磷酸并释放能量,形成的热力学拉力可促进合成反应的进行。球形红细菌(Rhodobacter sphaeroides 2.4.1)无机焦磷酸酶(RsPPase)属于II型可溶性焦磷酸酶,钴离子对于其活性的维持具有重要的功能,而其活性调控及其表达对细菌生长的影响尚未报道。研究克隆、原核表达纯化RsPPase。结果发现,钴离子会导致谷胱甘肽S-转移酶(glutathione sulfotransferase,GST)标签不能进行蛋白酶切,且融合蛋白GST-PPase水解焦磷酸的催化效率较低(Km/kcat=2.0×104mol(/L.s));在球形红细菌胞内表达大肠杆菌焦磷酸酶(Km/kcat=5.5×107mol/(L.s))没有影响细菌的生长,暗示球形红细菌胞内PPase活性足够高。通过对其结构的模拟推测,在钴离子存在的情况下为闭合构象,GST标签的存在可能影响PPase羧基端结构域的运动,而影响其结合底物和释放产物的能力;在钴离子不存在的情况下为开放构象,GST标签具有较大的自由度,可暴露蛋白酶识别位点酶切产生无标签RsPPase。

Prokaryotic Expression, Purification and Preliminary Analysis on Inorganic Pyrophosphatase from Rhodobacter sphaeroides

Inorganic pyrophosphatase(PPase) hydrolyzes pyrophosphate(PPi) generated from biosynthesis of macromolecules,such as protein,DNA,and fatty acids,provides the thermodynamic pull facilitating their biosynthesis.PPase from Rhodobacter sphaeroides(RsPPase) is one of the type II soluble PPase,and divalent cation Co2+ is essential for its activity.Regulation of RsPPase activity and effects of its expression on bacterium growth had not been reported yet.In this study,RsPPase was prokaryotically expressed and purified.It was found that fusion glutathione sulfotransferase tagged GST-PPase was non-cleavable by prescission protease in presence of Co2+,and had low catalytic efficiency(Km/kcat=2.0×104 mol/(L·s)).Little effect was found for R.sphaeroides growth by expressing PPase from E.coli(Km/kcat=5.5×107 mol/(L·s)),which indicated that intracellular RsPPase was enough for its growth.Based on the modeled structure,we speculated that in presence of Co2+ with the closed conformation,GST tag would restrict the movement of C terminal domain;therefore decrease the activity by affect the substrate binding and product release.In the absence of Co2+ with the open conformation,GST tag was more flexible,which made the protease recognition site available for protease to yield the non-tagged RsPPase.More work such as monomerization of RsPPase and extending the linker between GST tag and RsPPase,needs to be done to confirm this hypothesis.