Buhr A, Flükiger K, Erni B
Institute for Biochemistry, University of Bern, Switzerland.
J Biol Chem. 1994 Sep 23;269(38):23437-43.
The glucose transporter of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported sugar. It consists of a transmembrane subunit (IICBGlc) and a hydrophilic subunit (IIAGlc). The IICBGlc subunit consists of two domains. The NH2-terminal IIC domain (residues 1-386) spans the membrane eight times and contains the substrate binding site. The COOH-terminal hydrophilic IIB domain (residues 391-476) is accessible from the cytoplasmic side of the membrane. It contains the phosphorylation site (Cys421) and together with the IIC domain catalyzes the transfer of phosphoryl groups from the IIAGlc subunit to the transported solute. Starting from a plasmid vector containing ptsG under an inducible promoter, the IIB and the IIC domains have been subcloned separately, overexpressed in Escherichia coli, and purified by Ni2+ chelate affinity chromatography. Approximately 40 mg of IIBGlc-6H and 4 mg of IICGlc-6H could be purified from 1 liter of culture. Cells expressing IIBGlc-6H and IICGlc-6H separately have a three times longer generation time on glucose minimal medium than cells expressing wild-type IICBGlc. The rate of IIBGlc-6H phosphorylation determined in a nitrocellulose filter binding assay is indistinguishable from wild-type IICBGlc. The in vitro specific activity of IICGlc-6H in the presence of excess IIBGlc-6H is 2% of the control. IIBGlc-6H also complements the activity of a IICBGlc mutant with an inactive IIB domain (C421S) indicating that IIC and IIB are flexibly linked such that a free IIB domain can displace an inactive IIB domain from its contact site on the IIC domain. Based on this work, the secondary structure of the IIBGlc domain has been determined by isotope-edited NMR spectroscopy (Golic Grdadolnik, S., Eberstadt, M., Gemmecker, G., Kessler, H., Buhr, A., and Erni, B. (1994) Eur. J. Biochem. 219, 945-952).
细菌磷酸转移酶系统的葡萄糖转运蛋白将向量转运与被转运糖的磷酸化偶联起来。它由一个跨膜亚基（IICBGlc）和一个亲水性亚基（IIAGlc）组成。IICBGlc亚基由两个结构域组成。NH2末端的IIC结构域（第1 - 386位氨基酸残基）跨膜8次，包含底物结合位点。COOH末端的亲水性IIB结构域（第391 - 476位氨基酸残基）可从膜的细胞质一侧接触到。它包含磷酸化位点（Cys421），并与IIC结构域一起催化磷酸基团从IIAGlc亚基转移到被转运的溶质上。从一个在可诱导启动子控制下含有ptsG的质粒载体开始，IIB和IIC结构域已被分别亚克隆，在大肠杆菌中过量表达，并通过Ni2 +螯合亲和层析进行纯化。从1升培养物中大约可纯化出40 mg的IIBGlc - 6H和4 mg的IICGlc - 6H。在葡萄糖基本培养基上，分别表达IIBGlc - 6H和IICGlc - 6H的细胞的代时比表达野生型IICBGlc的细胞长三倍。在硝酸纤维素滤膜结合试验中测定的IIBGlc - 6H磷酸化速率与野生型IICBGlc没有区别。在过量IIBGlc - 6H存在下，IICGlc - 6H的体外比活性是对照的2%。IIBGlc - 6H也能补充具有无活性IIB结构域（C421S）的IICBGlc突变体的活性，这表明IIC和IIB是灵活连接的，使得一个游离的IIB结构域能够从其在IIC结构域上的接触位点取代一个无活性的IIB结构域。基于这项工作，IIBGlc结构域的二级结构已通过同位素编辑核磁共振光谱法确定（戈利奇·格拉多利尼克，S.，埃伯施塔特，M.，格梅克，G.，凯斯勒，H.，布尔，A.，和埃尔尼，B.（1994年）《欧洲生物化学杂志》219，945 - 952）。
