Chen Q, Amster-Choder O
Department of Molecular Biology, The Hebrew University-Hadassah Medical School, POB 12272, Jerusalem 91120, Israel.
Biochemistry. 1998 Dec 1;37(48):17040-7. doi: 10.1021/bi980067n.
The Escherichia coli BglF protein (EIIbgl) is an Enzyme II (EII) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) which catalyses transport and phosphorylation of beta-glucosides. In addition to its transport function, BglF serves as a beta-glucoside sensor which reversibly phosphorylates BglG, the transcription regulator of the bgl operon. Like many other PTS sugar permeases, the BglF protein is composed of three discrete functional and structural domains: IIAbgl and IIBbgl, which are hydrophilic, and IICbgl, which is hydrophobic. The domains of BglF are covalently linked to one another in the order BCA. The IIAbgl domain contains the first phosphorylation site, which accepts a phosphoryl group from the general PTS protein HPr and delivers it to the second phosphorylation site, located in the IIBbgl domain. This second site can deliver the phosphoryl group either to a beta-glucoside or to BglG. To elucidate the mechanism by which such different substrates can be phosphorylated by the same active site, we decided to try to separate the different phosphorylation activities catalyzed by BglF. To this end we rearranged the BglF domains and constructed IICBAbgl (scrambled-BglF). Scrambled-BglF behaved like wild-type BglF in its ability to be phosphorylated and to phosphorylate BglG in vitro and in vivo. However, it could not catalyze phosphorylation of beta-glucosides in vitro nor their phosphotransfer in vivo, and it could not catalyze BglG dephosphorylation in vitro or in vivo. Therefore, the two reactions induced by beta-glucosides, sugar phosphorylation and BglG dephosphorylation, seem to require a specific domain organization: IIBbgl should precede IICbgl. The order of the B and C domains is irrelevant for BglG phosphorylation, which occurs in the absence of beta-glucosides. Because the domain order affects the way that the domains are able to interact, our results suggest that catalysis of the sugar-induced functions depends on specific interactions between IIBbgl and IICbgl. In light of the previous assumption that domain order in EIIs is immaterial for their function, the finding that the order of the domains is important for the function of BglF as a sugar phosphotransferase raises two possibilities: (a) BglF differs from other EIIs in this regard; (b) BglF represents a subgroup of EIIs in which the requirement for a specific domain order correlates with the ability to transport a set of structurally related sugars.
大肠杆菌BglF蛋白（EIIbgl）是磷酸烯醇丙酮酸依赖性磷酸转移酶系统（PTS）的一种酶II（EII），它催化β-葡萄糖苷的转运和磷酸化。除了其转运功能外，BglF还作为一种β-葡萄糖苷传感器，可逆地磷酸化bgl操纵子的转录调节因子BglG。与许多其他PTS糖通透酶一样，BglF蛋白由三个离散的功能和结构域组成：亲水性的IIAbgl和IIBbgl，以及疏水性的IICbgl。BglF的结构域按BCA顺序彼此共价连接。IIAbgl结构域包含第一个磷酸化位点，它从通用PTS蛋白HPr接受一个磷酰基，并将其传递到位于IIBbgl结构域的第二个磷酸化位点。这个第二个位点可以将磷酰基传递给β-葡萄糖苷或BglG。为了阐明同一活性位点如何能磷酸化如此不同的底物的机制，我们决定尝试分离BglF催化的不同磷酸化活性。为此，我们重新排列了BglF结构域并构建了IICBAbgl（ scrambled-BglF）。在体外和体内，scrambled-BglF在被磷酸化以及磷酸化BglG的能力方面表现得与野生型BglF相似。然而，它在体外不能催化β-葡萄糖苷的磷酸化，在体内也不能催化其磷酸转移，并且在体外或体内都不能催化BglG的去磷酸化。因此，β-葡萄糖苷诱导的两个反应，即糖磷酸化和BglG去磷酸化，似乎需要特定的结构域组织：IIBbgl应先于IICbgl。B和C结构域的顺序对于在没有β-葡萄糖苷的情况下发生的BglG磷酸化是无关紧要的。由于结构域顺序影响结构域相互作用的方式，我们的结果表明糖诱导功能的催化取决于IIBbgl和IICbgl之间的特定相互作用。鉴于之前关于EIIs中结构域顺序对其功能无关紧要的假设，结构域顺序对BglF作为糖磷酸转移酶的功能很重要这一发现提出了两种可能性：（a）在这方面BglF与其他EIIs不同；（b）BglF代表EIIs的一个亚组，其中对特定结构域顺序的要求与转运一组结构相关糖的能力相关。
