Mapping the UDP-N-acetylglucosamine regulatory site of human glucosamine-6P synthase by saturation-transfer difference NMR and site-directed mutagenesis. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Biochimie Année : 2014

Mapping the UDP-N-acetylglucosamine regulatory site of human glucosamine-6P synthase by saturation-transfer difference NMR and site-directed mutagenesis.

Résumé

: The enzyme glucosamine-6P Synthase (Gfat, l-glutamine:d-fructose-6P amidotransferase) is involved in the hexosamine biosynthetic pathway and catalyzes the formation of glucosamine-6P from the substrates d-fructose-6-phosphate and l-glutamine. In eukaryotic cells, Gfat is inhibited by UDPGlcNAc, the end product of the biochemical pathway. In this work we present the dissection of the binding and inhibition properties of this feedback inhibitor and of its fragments by a combination of STD-NMR experiments and inhibition measurements on the wild type human enzyme (hGfat) as well as on site-directed mutants. We demonstrate that the UDPGlcNAc binding site is located in the isomerase domain of hGfat. Two amino acid residues (G445 and G461) located at the bottom of the binding site are identified to play a key role in the specificity of UDPGlcNAc inhibition of hGfat activity vs its bacterial Escherichia coli counterpart. We also show that UDPGlcNAc subcomponents have distinct features: the nucleotidic moiety is entirely responsible for binding whereas the N-acetyl group is mandatory for inhibition but not for binding, and the sugar moiety acts as a linker between the nucleotidic and N-acetyl groups. Combining these structural recognition determinants therefore appears as a promising strategy to selectively inhibit hGfat, which may for example help reduce complications in diabetes.
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Dates et versions

hal-00912794 , version 1 (02-12-2013)

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Nadine Assrir, Celine Richez, Philippe Durand, Eric Guittet, Bernard Badet, et al.. Mapping the UDP-N-acetylglucosamine regulatory site of human glucosamine-6P synthase by saturation-transfer difference NMR and site-directed mutagenesis.. Biochimie, 2014, 97, pp.39-48. ⟨10.1016/j.biochi.2013.09.011⟩. ⟨hal-00912794⟩
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