Asparagine 394 in putative helix 11 of the galactose-H+ symport protein (GalP) from Escherichia coli is associated with the internal binding site for cytochalasin B and sugar.

The Journal of Biological Chemistry
T P McDonaldP J Henderson

Abstract

The galactose-H+ symport protein (GalP) of Escherichia coli is very similar to the human glucose transport protein, GLUT1, and both contain a highly conserved Asn residue in predicted helix 11 that is different in a cytochalasin B-resistant member of this sugar transport family (XylE). The role of the Asn394 residue (which is predicted to be in putative trans-membrane alpha-helix 11) in the structure/activity relationship of the D-galactose-H+ symporter (GalP) was therefore assessed by measuring the interaction of sugar substrates and the inhibitory antibiotics, cytochalasin B, and forskolin with the wild-type and Asn394 --> Gln mutant proteins. Steady-state fluorescence quenching experiments show that the mutant protein binds cytochalasin B with a Kd 37-53-fold higher than the wild type. This low affinity binding was not detected with equilibrium binding or photolabeling experiments. In contrast, the mutant protein binds forskolin with a Kd similar to that of the wild type and is photolabeled by 3-125I-4-azido-phenethylamido-7-O-succinyl-desacetyl-forskolin. The mutant protein displays an increased amount of steady-state fluorescence quenching with the binding of forskolin, suggesting that the substitution of the Asn residue h...Continue Reading

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Citations

Aug 17, 2012·Applied Microbiology and Biotechnology·Jinlei TangXueli Zhang
Aug 28, 2012·Journal of Bacteriology·Sonja Steinsiek, Katja Bettenbrock
Sep 20, 2015·Metabolic Engineering·Shuang Ping LiuGui Yang Shi
Apr 15, 2016·Proceedings of the National Academy of Sciences of the United States of America·Khyati KapoorRobert M Stroud
Aug 17, 1999·Current Opinion in Cell Biology·M P BarrettG W Gould
Jun 28, 2016·Applied Microbiology and Biotechnology·Fernando Pérez-GarcíaVolker F Wendisch
Mar 10, 2015·Applied and Environmental Microbiology·Tian XiaMark A Eiteman
Jan 9, 2010·Rapid Communications in Mass Spectrometry : RCM·Lynsey N JonesAlison E Ashcroft
Aug 30, 2018·Microbial Cell Factories·L KyselovaK Bettenbrock
Aug 28, 2020·PLoS Computational Biology·John R Casey, Michael J Follows
Nov 18, 2018·Phytochemistry·Ronald StebeggAnnette Rompel

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