title: The glucose transporter of Escherichia coli with circularly permuted domains is active in vivo and in vitro.
abstract: The bacterial phosphotransferase system (PTS) consists of two energy-coupling soluble proteins (enzyme I and HPr) and a large number of inner membrane transporters (enzymes II) that mediate concomitant phosphorylation and translocation of sugars and hexitols. The transporters consist of three functional units (IIA, IIB, IIC), which occur either as protein subunits or domains of a multidomain polypeptide. The membrane-spanning IIC domain contains the substrate binding site; IIA and IIB are phosphorylation domains that transfer phosphate from HPr to the transported sugar. The transporter complexes of the PTS are good examples for variation of design by modular assembly of domains and subunits. The domain order is IIC-IIB in the membrane subunit of the Escherichia coli glucose transporter (IICBGlc) and IIB-IIC in Salmonella typhimurium sucrose transporter (IIBCScr). The phosphorylation domain of IICBGlc was translocated from the carboxyl-terminal to the amino-terminal end of the IIC domain, and the activity of the circularly permuted form was optimized by variation of the length and the composition of the interdomain linker. IIBapCGlc with an alanine-proline-rich interdomain linker has 70% of the control specific activity after purification and reconstitution into proteoliposomes. These results indicate that the amino-terminal end of IICBGlc must be on the cytoplasmic side of the inner membrane, that membrane insertion of the IIC domain is insensitive to the modification of its amino-terminal end, and that a domain swap as it could occur by a single DNA translocation event can rapidly lead to a functional protein. However, IIB could not be substituted for by glucokinase. Fusion proteins between the IIC domain and glucokinase do not transport and phosphorylate glucose in an ATP-dependent mechanism, although the IIC moiety displays transport activity upon complementation with soluble subclonal IIB, and the glucokinase moiety retains ATP-dependent nonvectorial kinase activity. This indicates that IIC and IIB are two cooperative units and not only sequentially acting upon a common substrate, and that translocation of glucose must be conformationally coupled to the phosphorylation/dephosphorylation cycle of IIB.
authors.affiliation: Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
authors.forename: R
authors.initials: R
authors.lastname: Gutknecht
authors.forename: M
authors.initials: M
authors.lastname: Manni
authors.forename: Q
authors.initials: Q
authors.lastname: Mao
authors.forename: B
authors.initials: B
authors.lastname: Erni
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001692:Biological Transport / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism*; D005941:Glucokinase / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D009154:Mutation / Q000235:genetics; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation; D010770:Phosphotransferases / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D010770:Phosphotransferases; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D005941:Glucokinase