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 (PTS) consists of a hydrophilic (IIAGlc) and a transmembrane subunit (IICBGlc). IICBGlc has two domains (C and B), which are linked by a highly 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 arginines (Arg-424 and Arg-426) adjacent to the phosphorylation site (Cys-421), the invariant interdomain sequence KTPGRED, and two conserved histidines (His-211 and His-212) in the IIC domain were mutated and the mutant proteins characterized in vivo and in vitro for transport and phosphorylation activity. Replacement of the strongly beta-turn favoring residues Thr and Gly of the linker by alpha-helix favoring Ala results in strong reduction of activity, whereas the substitutions of the other residues have only minor effects. The R424K and R426K mutants can be phosphorylated by IIAGlc but can no longer donate the phosphoryl group to glucose. The H211Q and H212Q mutants continue to phosphorylate glucose at a reduced rate but H212Q can no longer transport glucose. Mixtures of purified R424K/H212Q and R426K/H212Q have 10% of wild-type phosphorylation activity and when coexpressed in Escherichia coli support glucose transport.
Lanz
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R
Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012, Bern, Switzerland.
Erni
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B
D000409:Alanine; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D009004:Monosaccharide Transport Proteins; D006639:Histidine; D001120:Arginine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D000409:Alanine