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, r espectively, in Klebsiella pneumoniae 1033-5P14, derivative KAY2026. E ach gene codes for a specific transporter which, based on sequence dat a, belongs to a large family of carbohydrate transporters which consti tutes 12 transmembrane helices, DalT and RbtT show an unusually high s imilarity (86.2% identical residues for totals of 425 and 427 amino ac ids, respectively). This allowed the construction of DalT'-Rbt'T and R bt-T'-Dal'T crossover hybrids by using a natural restriction site over lapping Met202, This site is located within the large cytoplasmic loop which connects the putative helices 6 and 7 and in particular the ami no-and the carboxy-terminal halves of the transporters, Both hybrids h ave 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 import-ant class of carbohydrate transporters.