Will, Andreas and Grassl, Renate and Erdmenger, Jörg and Caspari, Thomas and Tanner, Widmar (1998) Alteration of substrate affinities and specificities of the Chlorella hexose/H+ symporters by mutations and construction of chimeras. JOURNAL OF BIOLOGICAL CHEMISTRY, 273 (19). pp. 11456-11462. ISSN 0021-9258, 1083-351X
Full text not available from this repository.Abstract
The cDNAs HUP1 and HUP2 of Chlorella kessleri code for monosaccharide/H+ symporters that can be functionally expressed in Schizosaccharomyces pombe, By random mutagenesis three HUP1 mutants with an increased K-m value for D-glucose were isolated. The 40-fold increase in K-m of the first mutant is due to the amino acid exchange N436I in putative transmembrane helix XI, Two substitutions were found in a second (G97C/I303N) and third mutant (G120D/F292L), which show a 270-fold and 50-fold increase in K-m for D-glucose, respectively, An investigation of the individual mutations revealed that the substitutions I303N and F292L (both in helix VII) cause the K-m shifts seen in the corresponding double mutants. These mutations together with those previously found support the hypothesis that helices V, VII, and XI participate in the transmembrane sugar pathway. Whereas for most mutants obtained so far the K-m change for D-glucose is paralleled by a corresponding change for other hexoses tested, the exchange D44E exclusively alters the K-m for D-glucose, Moreover the pH profile of this mutant is shifted by more than 2 pH units to alkaline values, indicating that the activity of the transporter may require deprotonation of the corresponding carboxyl group. Chimeric transporters were constructed to study the 100-fold lower affinity for D-galactose of the HUP1 symporter as compared with that of the HUP2 protein. A crucial determinant for the differential D-galactose recognition was shown to be associated with the first external loop. The effect could be pinpointed to a single amino acid change: replacement of Asn-45 of HUP1 with isoleucine, the corresponding amino acid of HUP2, yields a transporter with a 20 times higher affinity for D-galactose, The reverse substitution (I47N) decreases the affinity of HUP2 for D-galactose 20-fold.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | YEAST SACCHAROMYCES-CEREVISIAE; GLUT1 GLUCOSE-TRANSPORTER; EXOFACIAL LIGAND-BINDING; ESCHERICHIA-COLI; SCHIZOSACCHAROMYCES-POMBE; HETEROLOGOUS EXPRESSION; H+-COTRANSPORTER; SUGAR-TRANSPORT; FISSION YEAST; RECOGNITION |
| Subjects: | 500 Science > 570 Life sciences 500 Science > 580 Botanical sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Pflanzenwissenschaften > Lehrstuhl für Zellbiologie und Pflanzenphysiologie (Prof. Dr. Klaus Grasser) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 24 Feb 2023 08:03 |
| Last Modified: | 24 Feb 2023 08:03 |
| URI: | https://pred.uni-regensburg.de/id/eprint/49814 |
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