Mutational analysis of invariant arginines in the IIAB(Man) subunit of the Escherichia coli phosphotransferase system.
The mannose transporter of bacterial phosphotransferase system mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. It consists of the transmembrane IIC(Man)-IID(Man) complex and the cytoplasmic IIAB(Man) subunit. IIAB(Man) has two flexibly linked domains, IIA(Man) and IIB(Man), each containing a phosphorylation site (His-10 and His-175). Phosphoryl groups are transferred from the phosphoryl carrier protein phospho-HPr to His-10, hence to His-175 and finally to the 6' OH of the transported hexose. Phosphate-binding sites and phosphate-catalytic sites frequently contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions. IIB(Man) contains five arginines which are invariant in the homologous IIB subunits of Escherichia coli, Klebsiella pneumoniae and Bacillus subtilis. The IIA domains have no conserved arginines. The five arginines were replaced by Lys or Gln one at a time, and the mutants were analyzed for transport and phosphorylation activity. All five IIB mutants can still be phosphorylated at His-175 by the IIA domain. R172Q is completely inactive with respect to glucose phosphotransferase (phosphoryltransfer from His-175 to the 6' OH of Glc) and hexose transport activity. R168Q has no hexose transport and strongly reduced phosphotransferase activity. R204K has no transport but almost normal phosphotransferase activity. R304Q has only slightly reduced transport activity. R190K behaves like wild-type IIAB(Man). Arg-168, Arg-172, and Arg-304 are part of the hydrogen bonding network on the surface of IIB, which contains the active site His-175 and the interface with the IIA domain (Schauder, S., Nunn, R.S., Lanz, R., Erni, B. and Schirmer, T. (1998) J. Mol. Biol. 276, 591-602) (Protein Data Bank accession code 1BLE). Arg-204 is at the putative interface between IIB(Man) and the IIC(Man)-IID(Man) complex.
Gutknecht
R
R
Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012, Bern, Switzerland.
Lanz
R
R
Erni
B
B
D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D004926:Escherichia coli / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D017510:Protein Folding; D014961:X-Ray Diffraction
D001120:Arginine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C037992:phosphoenolpyruvate-mannose phosphotransferase