Substrate recognition domains as revealed by active hybrids between the D-arabinitol and ribitol transporters from Klebsiella pneumoniae

Two new genes, dalT and rbtT, have been cloned from the dal operon for D-arabinitol and the rbt operon for ribitol uptake and degradation, respectively, in Klebsiella pneumoniae 1033-5P14, derivative KAY2026. Each gene codes for a specific transporter which, based on sequence data, belongs to a large family of carbohydrate transporters which constitutes 12 transmembrane helices. DalT and RbtT show an unusually high similarity (86.2% identical residues for totals of 425 and 427 amino acids, respectively). This allowed the construction of DalT'-Rbt"T and RbtT'-Dal'T crossover hybrids by using a natural restriction site overlapping Met202. This site is located within the large cytoplasmic loop which connects the putative helices 6 and 7 and in particular the amino- and the carboxy-terminal halves of the transporters. Both hybrids have close to normal transport activities but essentially the substrate specificities and kinetic properties of the amino-terminal half. This result localizes essential substrate binding and recognition sites to the amino-terminal halves of the proteins in this important class of carbohydrate transporters.