Mapping the UDP-N-acetylglucosamine regulatory site of human glucosamine-6P synthase by saturation-transfer difference NMR and site-directed mutagenesis.
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.
Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, UPR2301, CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Nadine
N
Assrir
Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, UPR2301, CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Celine
C
Richez
Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, UPR2301, CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Philippe
P
Durand
Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, UPR2301, CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Eric
E
Guittet
Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, UPR2301, CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Bernard
B
Badet
Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, UPR2301, CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France. Electronic address: ewen.lescop@cnrs.fr.
Ewen
E
Lescop
Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, UPR2301, CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France. Electronic address: marie-ange.badet@cnrs.fr.
Marie-Ange
MA
Badet-Denisot
D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D025461:Feedback, Physiological; D005636:Fructosephosphates / Q000737:chemistry / Q000378:metabolism*; D015870:Gene Expression; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000737:chemistry / Q000378:metabolism*
D005636:Fructosephosphates; D011994:Recombinant Proteins; D005973:Glutamine; C001293:glucosamine 6-phosphate; D014537:Uridine Diphosphate N-Acetylglucosamine; D019298:Glucose-6-Phosphate; C027618:fructose-6-phosphate; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005944:Glucosamine
Allosteric regulation; Fru6P; Glc6P; GlcN6P; Glucosamine-6-phosphate synthase; Glutamine amidotransferase; NMR; STD; STD-NMR; TCEP; Transferred NOE; Tris(2-carboxyethyl)phosphine hydrochloride; UDP-N-acetyl-d-glucosamine; UDPGlcNAc; UDPGlcNAc binding site; d-fructose-6-phosphate; d-glucosamine-6-phosphate; d-glucose-6-phosphate; hGfat; human l-glutamine:d-fructose-6P amidotransferase or glucosamine-6-phosphate synthase; nuclear magnetic resonance; saturation transfer difference