The different functions of BglF, the E-coli beta-glucoside permease and sensor of the bgl system, have different structural requirements

The Escherichia coli BglF protein (EIIbgl) is an Enzyme II (EII) of the pho sphoenolpyruvate dependent phosphotransferase system (PTS) which catalyses transport and phosphorylation of beta-glucosides. In addition to its transp ort function, BglF serves as a beta-glucoside sensor which reversibly phosp horylates BglG, the transcription regulator of the bgl operon. Like many ot her PTS sugar permeases, the BglF protein is composed of three discrete fun ctional and structural domains: IIA(bgl) and IIBbgl, which are hydrophilic, and IICbgl, which is hydrophobic. The domains of BglF are covalently linke d to one another in the order BCA. The IIAbgl domain contains the first pho sphorylation site, which accepts a phosphoryl group from the general PTS pr otein HPr and delivers it to the second phosphorylation site, located in th e IIBbgl domain. This second site can deliver the phosphoryl group either t o a beta-glucoside or to BglG. To elucidate the mechanism by which such dif ferent substrates can be phosphorylated by the same active site, we decided to try to separate the different phosphorylation activities catalyzed by B glF. To this end we rearranged the BglF domains and constructed IICBA(bgl) (scrambled-BglF). Scrambled-BglF behaved like wild-type BglF in its ability to be phosphorylated and to phosphorylate BglG in vitro and in vivo. Howev er, it could not catalyze phosphorylation of beta-glucosides in vitro nor t heir phosphotransfer in vivo, and it could not catalyze BglG dephosphorylat ion in vitro or in vivo. Therefore, the two reactions induced by beta-gluco sides, sugar phosphorylation and BglG dephosphorylation, seem to require a specific domain organization: IIBbgl should precede IICbgl. The order of th e B and C domains is irrelevant for BglG phosphorylation, which occurs in t he absence of beta-glucosides. Because the domain order affects the way tha t the domains are able to interact, our results suggest that catalysis of t he sugar-induced functions depends on specific interactions between IIBbgl and IICbgl. In light of the previous assumption that domain order in EIIs i s 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) Bgl F represents a subgroup of EIIs in which the requirement for a specific dom ain order correlates with the ability to transport a set of structurally re lated sugars.