glpX Gene of Mycobacterium tuberculosis: Heterologous Expression, Purification, and Enzymatic Characterization of the Encoded Fructose 1,6-bisphosphatase II
glpX Gene of Mycobacterium tuberculosis: Heterologous Expression, Purification, and Enzymatic Characterization of the Encoded Fructose 1,6-bisphosphatase II
复制标题
DOI：
10.1007/s12010-011-9219-x
复制
发表时间：
2011-08-01
影响因子：
3
通讯作者：
Movahedzadeh, Farahnaz
中科院分区：
文献类型：
--
作者：
Gutka, Hiten J.;Rukseree, Kamolchanok;Movahedzadeh, Farahnaz
结核分枝杆菌（Mtb）的glpX基因（Rv 1099 c）编码果糖1，6-二磷酸酶II（FBPase II; EC 3.1.3.11）;一种关键的致突变酶。Mtb具有glpX同源物作为已知的主要FBPase。本研究探讨了结核分枝杆菌FBPase II的表达、纯化和酶学性质。glpX基因的克隆，表达和纯化使用两步纯化策略，包括亲和和尺寸排阻色谱。Mtb FBPase II的比活性为1.3 U/mg。该酶是寡聚的，遵循Michaelis-Menten动力学，表观km = 44 μ M。酶活性依赖于二价金属离子，并被锂和无机磷酸盐抑制。测定了该酶的最适pH值和热稳定性。稳健的表达、纯化和测定方案确保了该蛋白质的充分生产，用于结构生物学和针对该酶的抑制剂的筛选。
The glpX gene (Rv1099c) of Mycobacterium tuberculosis (Mtb) encodes Fructose 1,6-bisphosphatase II (FBPase II; EC 3.1.3.11); a key gluconeogenic enzyme. Mtb possesses glpX homologue as the major known FBPase. This study explored the expression, purification and enzymatic characterization of functionally active FBPase II from Mtb. The glpX gene was cloned, expressed and purified using a two step purification strategy including affinity and size exclusion chromatography. The specific activity of Mtb FBPase II is 1.3 U/mg. The enzyme is oligomeric, followed Michaelis-Menten kinetics with an apparent km = 44 mu M. Enzyme activity is dependent on bivalent metal ions and is inhibited by lithium and inorganic phosphate. The pH optimum and thermostability of the enzyme have been determined. The robust expression, purification and assay protocols ensure sufficient production of this protein for structural biology and screening of inhibitors against this enzyme.