MUTATIONAL ANALYSIS OF INVARIANT ARGININES IN THE IIAB(MAN) SUBUNIT OF THE ESCHERICHIA-COLI PHOSPHOTRANSFERASE SYSTEM

The mannose transporter of bacterial phosphotransferase system mediate s uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. It consis ts of the transmembrane IICMan-IIDMan complex and the cytoplasmic IIAB (Man) subunit, IIAB(Man) has two flexibly linked domains, IIA(Man) and IIBMan, 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 th e 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 intera ctions. IIBMan contains five arginines which are invariant in the homo logous IIB subunits of Escherichia coli, Klebsiella pneumoniae and Bac illus subtilis, The IIA domains have no conserved arginines, The five arginines were replaced by Lys or Gin one at a time, and the mutants w ere analyzed for transport and phosphorylation activity. All five HB m utants can still be phosphorylated at His-175 by the IIA domain. R172Q is completely inactive with respect to glucose phosphotransferase (ph osphoryltransfer from His-175 to the 6' OH of Glc) and hexose transpor t activity. R168Q has no hexose transport and strongly reduced phospho transferase activity. R204K, has no transport but almost normal phosph otransferase activity, R304Q has only slightly reduced transport activ ity. 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 HB, wh ich contains the active site His-175 and the interface with the LIA do main (Schauder, S., Nunn, R.S., Lanz, R., Erni, B. and Schirmer, T. (1 998) J. Mol. Biol, 276, 591-602) (Protein Data Bank accession code 1BL E), Arg-204 is at the putative interface between IIBMan and the IICMan -IIDMan complex.