Research paper Mapping the UDP-N-acetylglucosamine regulatory site of human glucosamine-6P synthase by saturation-transfer difference NMR and site-directed mutagenesis Nadine Assrir, Celine Richez, Philippe Durand, Eric Guittet, Bernard Badet, Ewen Lescop ** , Marie-Ange Badet-Denisot * Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, UPR2301, CNRS,1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France article info Article history: Received 12 June 2013 Accepted 13 September 2013 Available online 26 September 2013 Keywords: Glucosamine-6-phosphate synthase UDPGlcNAc binding site Glutamine amidotransferase STD-NMR Allosteric regulation Transferred NOE abstract 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 sub- strates 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 ex- periments 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 in- hibition 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. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction Human glucosamine-6P synthase (hGfat) catalyzes the rate- limiting step of the hexosamine biosynthetic pathway leading to the production of UDP-N-acetyl-D-glucosamine (UDPGlcNAc, see Fig. 1). The latter is used as a substrate by the N-acetylglucosaminyl transferase (OGT) to install the acetylated amino-sugar on target proteins. Protein O-GlcNAcylation is involved in many cellular processes including signal transduction, protein trafficking, protein degradation and regulation of gene expression as mentioned in recent reviews [1,2]. O-glycosylation and phosphorylation can compete at the same amino-acid positions suggesting a complex interplay between phosphorylation and O-GlcNAcylation [3]. The hexosamine biosynthetic pathway is assumed to play a role in diabetes and glucose toxicity [4e8]. Indeed the conversion of glucose to glucosamine-6P is linked to glucose-induced insulin resistance as showed by studies in 3T3 L1 adipocytes [9] and earlier works on Gfat [10e12]. The emerging role of GlcNAcylation in cellular signaling and diseases prompted us to investigate the regulation mechanism of hGfat, which controls the glucosamine-6P supply required for such a modification [13,14]. Each monomer of this tetrameric enzyme (4  77.6 kDa) con- tains two catalytic domains in charge of binding the substrates L- glutamine (glutaminase domain) and D-fructose-6P (isomerase domain) respectively [15]. The cysteine1-mediated hydrolysis of glutamine carboxamide group first occurs at the glutaminase site. The formed ammonia is then transferred to the isomerase domain where condensation with fructose-6P (Fru6P) takes place before ketimine/aldimine isomerization and glucosamine-6P (GlcN6P) release (Scheme 1) [16]. As a ubiquitous protein, the enzyme ex- hibits high sequence homology among different species: for example, human hGfat shares 99, 84 and 69% sequence homology Abbreviations: Fru6P, D-fructose-6-phosphate; Glc6P, D-glucose-6-phosphate; GlcN6P, D-glucosamine-6-phosphate; hGfat, human L-glutamine:D-fructose-6P amidotransferase or glucosamine-6-phosphate synthase; NMR, nuclear magnetic resonance; STD, saturation transfer difference; TCEP, Tris(2-carboxyethyl)phos- phine hydrochloride; UDPGlcNAc, UDP-N-acetyl-D-glucosamine. * Corresponding author. Tel.: þ33 1 69 82 31 10; fax: þ33 169 82 72 47. ** Corresponding author. Tel.: þ33 1 69 82 37 64; fax: þ33 1 69 82 37 84. E-mail addresses: ewen.lescop@cnrs.fr (E. Lescop), marie-ange.badet@cnrs.fr (M.-A. Badet-Denisot). Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.09.011 Biochimie 97 (2014) 39e48