THE GLUCOSE-TRANSPORTER OF THE ESCHERICHIA-COLI PHOSPHOTRANSFERASE SYSTEM - MUTANT ANALYSIS OF THE INVARIANT ARGININES, HISTIDINES, AND DOMAIN LINKER

The glucose transporter of the bacterial phosphotransferase system (PT S) consists of a hydrophilic (IIA(Glc)) and a transmembrane subunit (I ICBGlc). IICBGlc has two domains (C and B), which are linked by a high ly invariant sequence. Transport of glucose by IIC and phosphorylation by IIB are tightly coupled processes. Three motifs that are strongly conserved in 12 homologous PTS transporters, namely two invariant argi nines (Arg-424 and Arg-426) adjacent to the phosphorylation site (Cys- 421), the invariant interdomain sequence KTPGRED, and two conserved hi stidines (His-211 and His-212) in the IIC domain were mutated and the mutant proteins characterized in vivo and in vitro for transport and p hosphorylation activity. Replacement of the strongly beta-turn favorin g residues Thr and Gly of the linker by alpha-helix favoring Ala resul ts in strong reduction of activity, whereas the substitutions of the o ther residues have only minor effects. The R424K and R426K mutants can be phosphorylated by IIA(Glc) but can no longer donate the phosphoryl group to glucose. The H211Q and H212Q mutants continue to phosphoryla te glucose at a reduced rate but H212Q can no longer transport glucose . Mixtures of purified R424K/H212Q and R426K/H212Q have 10% of wild-ty pe phosphorylation activity and when coexpressed in Escherichia coil s upport glucose transport.