SUBUNIT AND AMINO-ACID INTERACTIONS IN THE ESCHERICHIA-COLI MANNITOL PERMEASE - A FUNCTIONAL COMPLEMENTATION STUDY OF COEXPRESSED MUTANT PERMEASE PROTEINS

Mannitol-specific enzyme II, or mannitol permease, of the phosphaenolp yruvate-dependent carbohydrate phosphotransferase system of Escherichi a coli carries out the transport and phosphorylation of D-mannitol and is most active as a dimer in the membrane. We recently reported the i mportance of a glutamate residue at position 257 in the binding and tr ansport of mannitol by this protein (C. Saraceni-Richards and G. R. Ja cobson, J. Bacteriol. 179:1135-1142, 1997). Replacing Glu-257 with ala nine (E257A) or glutamine (E257Q) eliminated detectable mannitol bindi ng and transport by the permease, In contrast, an E257D mutant protein was able to bind and phosphorylate mannitol in a manner similar to th at of the wild-type protein but was severely defective in mannitol upt ake, In this study, me have coexpressed proteins containing mutations at position 257 with other inactive permeases containing mutations in each of the three domains of this protein, Activities of any active he terodimers resulting from this coexpression were measured. The results show that various inactive mutant permease proteins can complement pr oteins containing mutations at position 257, In addition, we show that both Glu at position 257 and His at position 195, both of which are i n the membrane-bound C domain of the protein, must be on the same subu nit of a permease dimer in order for efficient mannitol phosphorylatio n and uptake to occur, The results also suggest that mannitol bound to the opposite subunit within a permease heterodimer can be phosphoryla ted by the subunit containing the E257A mutation (which cannot bind ma nnitol) and support a model in which there are separate binding sites on each subunit within a permease dimer, Finally, we provide evidence from these studies that high-affinity mannitol binding is necessary fo r efficient transport by mannitol permease.