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.
Heuel
H
H
Universität Osnabrück, Fachbereich Biologie/Chemie, AG Genetik, Germany.
Turgut
S
S
Schmid
K
K
Lengeler
J W
JW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012255:Ribitol / Q000378:metabolism*; D013379:Substrate Specificity; D013402:Sugar Alcohols / Q000378:metabolism*; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D002352:Carrier Proteins; C108446:DalT protein, Klebsiella pneumoniae; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C108447:RbtT protein, Klebsiella pneumoniae; D011993:Recombinant Fusion Proteins; D013402:Sugar Alcohols; D012255:Ribitol; C014999:arabitol