THE MANNOSE TRANSPORTER OF ESCHERICHIA-COLI - STRUCTURE AND FUNCTION OF THE IIAB(MAN)-SUBUNIT

The mannose transporter of the bacterial phosphotransferase system con sists of two transmembrane subunits (IIC(Man) and IID(Man)) and a hydr ophilic subunit (IIAB(Man)). IIAB(Man) has two flexibly linked domains containing one phosphorylation site each and occurs as a dimer. Subst rate transport is coupled to phosphorylation. The phosphoryl group is transferred from a phosphoryl carrier protein to His10 on IIA, hence t o His175 on IIB and finally to the substrate. IIAB(Man) mutants were a nalyzed in vitro for complementation, negative dominance, cysteine cro ss-linking and reactivity. Conclusions: (i) His10, Trp12, Lys48, and S er72 form a functional unit (phosphorylation site 1); (ii) His86 on th e IIA domain and His175 on the IIB domain of the same subunit form a f unctional unit (phosphorylation site 2); (iii) phosphoryl transfer can occur between His10 and His86 of the same as well as of different sub units and His86 is necessary for this transfer; (iv) the subunits in t he dimer are interdependent; (v) The phosphorylation site mutant H175C is highly reactive toward thiol reagents and it forms extensive homo- and heterocross-links with other surface-exposed cysteines. The phosp horylation site mutant H10C is 1000-fold less reactive. The two residu es might be in complementary locations, His10 buried in a concave, His 175 exposed on a convex surface.