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Glutamine Binding Opens the Ammonia Channel and Activates Glucosamine-6P Synthase*

Glucosamine-6P synthase catalyzes the synthesis of glucosamine-6P from fructose-6P and glutamine and uses a channel to transfer ammonia from its glutaminase to its synthase active site. X-ray structures of glucosamine-6P synthase have been determined at 2.05 Å resolution in the presence of fructose-6P and at 2.35 Å resolution in the presence of fructose-6P and 6-diazo-5-oxo-l-norleucine, a glutamine affinity analog that covalently modifies the N-terminal catalytic cysteine, therefore mimicking the γ-glutamyl-thioester intermediate formed during hydrolysis of glutamine. The fixation of the glutamine analog activates the enzyme through several major structural changes: 1) the closure of a loop to shield the glutaminase site accompanied by significant domain hinging, 2) the activation of catalytic residues involved in glutamine hydrolysis, i.e. the α-amino group of Cys-1 and Asn-98 that is positioned to form the oxyanion hole, and 3) a 75° rotation of the Trp-74 indole group that opens the ammonia channel.

Glucosamine-6P synthase catalyzes the synthesis of glucosamine-6P from fructose-6P and glutamine and uses a channel to transfer ammonia from its glutaminase to its synthase active site. X-ray structures of glucosamine-6P synthase have been determined at 2.05 Å resolution in the presence of fructose-6P and at 2.35 Å resolution in the presence of fructose-6P and 6-diazo-5-oxo-L-norleucine, a glutamine affinity analog that covalently modifies the N-terminal catalytic cysteine, therefore mimicking the ␥-glutamylthioester intermediate formed during hydrolysis of glutamine. The fixation of the glutamine analog activates the enzyme through several major structural changes: 1) the closure of a loop to shield the glutaminase site accompanied by significant domain hinging, 2) the activation of catalytic residues involved in glutamine hydrolysis, i.e. the ␣-amino group of Cys-1 and Asn-98 that is positioned to form the oxyanion hole, and 3) a 75°rotation of the Trp-74 indole group that opens the ammonia channel.
Glucosamine-6P synthase (GlmS) 2 catalyzes the first and rate-limiting step of hexosamine metabolism, the conversion of D-fructose-6P (Fru6P) into D-glucosamine-6P using L-glutamine as a nitrogen source. Because the end product of this pathway, UDP-N-acetyl glucosamine, is a major building block of the bacterial peptidoglycan and fungal chitin, inhibitors of the microbial enzymes have potential antifungal and antibiotics properties. More recently, the human enzyme, Gfat, has attracted significant attention because of the observation that hexosamine biosynthetic pathway plays a nutrient sensor role and is a mediator of insulin resistance (1). Indeed, transgenic mice overexpressing glucosamine 6-phosphate synthase in skeletal muscle and fat have been shown to develop insulin resistance with manifestations similar to that observed in type-2 diabetes (2). Therefore, inhibitors directed toward the human enzyme are expected to limit the renal and ocular complications associated with type-2 diabetes in diabetes treatment. Understanding at the molecular level the particular mechanisms of the different enzymes is essential to development of specific drugs against the human, fungal, or bacterial enzymes. GlmS belongs to the glutamine amidotransferase family that uses the nucleophilic attack of the thiol group of a cysteine residue on the ␦-carbonyl group of glutamine to form ammonia, which is next transferred to an NH 3 acceptor substrate that differs for each amidotransferase (3,4). Glutamine amidotransferases provide the major route for incorporation of nitrogen into the biosynthetic pathways of amino acids, amino sugars, purine and pyrimidine nucleotides, coenzymes, and antibiotics. Together with glutamate synthase, glutamine phosphoribosylpyrophosphate amidotransferase, and asparagine synthetase B, GlmS belongs to the N-terminal nucleophile (Ntn) subclass of amidotransferases that uses the ␣-amino group of the terminal nucleophilic cysteine residue to activate its thiol group (5). GlmS from Escherichia coli consists of a 27-kDa glutaminase domain ( N GlmS, residues 1-239) that catalyzes glutamine hydrolysis and a 40-kDa synthase domain ( C GlmS, residues 249 -608) that catalyzes Fru6P amination and isomerization ( Fig. 1) (6, 7). The x-ray structures of the individual domains have been previously determined for the glutaminase domain in the presence of L-glutamate or ␥-glutamyl hydroxamate (8) and for the synthase domain in the presence of glucosamine-6P, glucose-6P (Glc6P), or 2-amino-deoxyglucitol-6P (9, 10). The binding site residues were identified, and a mechanism at each active site was proposed ( Fig. 1) in agreement with the biochemical data (11). The structures also revealed the homodimeric nature of the enzyme but could not give any information about the communication between the two domains. The next crystal structure at 3.1 Å resolution of the whole enzyme crystallized in the presence of Fru6P showed the existence of an 18 Å-long channel connecting the glutaminase and isomerase sites (12). The walls of the ammonia channel were constituted by the side-chain of Trp-74 and the main-chain of Arg-26, both from the glutaminase domain, and by residues 601-607 belonging to the C-terminal nonapeptide (C-tail, residues 600 -608), as well as a loop of the synthase domain of the neighboring subunit (residues 503*-505*). Unexpectedly, the channel was totally blocked by the Trp-74 indole group. This provided a clue to explain why GlmS, together with glutamate synthase, are the only amidotransferases that cannot use exogenous ammonia as a nitrogen donor instead of glutamine (13,14).
The solvent-inaccessible ammonia channel in the amidotransferase family provides a great advantage for catalysis as it prevents the formation of non-reactive ammonium ions and the loss of the reaction intermediates in solution. To achieve this goal, the enzymes must synchronize their catalytic sites through large domain movements triggered by substrate binding (15)(16)(17). The different corresponding conformational states of the proteins can be visualized by the crystal structures of the enzymes in the presence of various ligands. GlmS uses an ordered bi-bi mechanism with Fru6P binding prior to glutamine (18). A model for substrate binding and enzyme activation through conformational changes has been proposed based on the crystal structure at 3.1 Å resolution of GlmS in complex with the sugar (12). The rearrangement of the C-tail occurring upon Fru6P binding would close the synthase site and activate the glutaminase function. This scheme is supported by the half-of-the-site reactivity behavior of GlmS for affinity labeling in the absence of Fru6P (18). However, this conformational change could be visualized only by comparing the structures of GlmS in the absence of the substrates and in the presence of Fru6P alone. Second, glutamine binding would trigger another conformational change that would result in the closure of the glutaminase site by a flexible loop from the glutaminase domain (glutamine loop or Q-loop, residues 73-81) and the opening of the channel.
To verify this assumption and get a better understanding of the structural communication between the two active sites occurring upon glutamine binding, the crystal structure of GlmS at 2.05 Å resolution in the presence of Fru6P and the structure at 2.35 Å resolution in the presence of both Fru6P and the glutamine affinity analog 6-diazo-5-oxo-L-norleucine (DON) were determined and compared. The DON inhibitor is known to irreversibly alkylate the thiol group of the N-terminal cysteine in Ntn amidotransferases, and the resulting alkyl-enzyme 9 mimics the putative covalent ␥-glutamyl-thioester intermediate 2 formed during glutamine hydrolysis (19, 20) (Fig. 1).

EXPERIMENTAL PROCEDURES
Protein Preparation, Purification, and Crystallization-GlmS was purified from recombinant E. coli HB101 cells at 4°C. After growth at 37°C under strong aeration during 17 h in 4 liters of Luria Bertani medium supplemented with 100 g ml Ϫ1 ampicillin, cells were resuspended in 100 ml of buffer A (20 mM Bis Tris Propane, pH 7.2, 1 mM EDTA, 1 mM dithiothreitol) and sonicated. The supernatant was loaded onto a Q-Sepharose fast flow column (5 ϫ 15 cm) equilibrated in buffer A. The column was washed and eluted with a linear gradient of 0 -1 M NaCl in buffer A. Active fractions eluting at 0.35 M NaCl were pooled, concentrated, and loaded onto a Superdex 200 Hiload gel filtration column previously equilibrated in 20 mM KPO 4 , pH 7.2. The active fractions (972 mg) were pooled and concentrated.
The DON-inactivated GlmS was formed by incubating GlmS with 2 mM DON and 10 mM Fru6P for 1 h in 50 mM KPO 4 , pH 7.2, loaded onto a Mono Q HR 10/10 column, and eluted with a linear gradient of 0 -1 M NaCl in 10 mM Fru6P, 20 mM HEPES, pH 7.2. Crystals were grown at FIGURE 1. A, catalytic mechanism at the synthase domain. The opening of Fru6P triggered by His-504* is followed by the formation of the Schiff base 2 between Lys-603 and the carbonyl group of linear Fru6P 1. Transimination with ammonia from the glutaminase site yields fructosimine-6P 3. Abstraction of its C1 proR proton by Glu-488 produces the cis-enolamine 4, which is reprotonated at C2 to yield the linear form of glucosamine-6P 5 that is closed by His-504*. B, catalytic mechanism at the glutaminase domain. The N-terminal group activates the thiol group of Cys-1 for nucleophilic attack on the carboxamide group of glutamine, producing an oxyanion tetrahedral intermediate 6, stabilized by N␦2 of Asn-98 and NH of Gly-99, that breaks down to yield ␥-glutamylthioester 7 and ammonia. Hydrolysis of 7 proceeds through the oxyanion 8, producing glutamate and free enzyme. The N-terminal group is therefore supposed to act alternatively as a general base in the activation of the nucleophilic Cys-1 and in the hydrolysis of ␥-glutamyl-thioester 7 and as a general acid in the protonation of N␦2 of glutamine to form ammonia. C, inactivation of GlmS by DON. DON activation results from inhibitor protonation, possibly by the Cys-1 thiol group. The following nucleophilic attack of the thiolate group on the diazonium group of the inhibitor leads to alkyl-enzyme 4, which mimics ␥-glutamyl-thioester 2.
X-ray Data Collection and Processing-Crystals were soaked for a few minutes in a cryoprotectant solution (25% glycerol, 16% polyethylene glycol 8000, 0.1 M KCl for the GlmS⅐Glc6P⅐DON crystal and 25% polyethylene glycol 400, 0.1 M HEPES, pH 7.5, 1 M LiCl for the GlmS⅐Fru6P crystal) before flash freezing in a cold nitrogen stream at 100 K. The diffraction data sets were collected on beam line ID14-EH2 for the GlmS⅐Glc6P⅐DON crystal or ID14-EH1 for the GlmS⅐Fru6P crystal at the European Synchrotron Radiation Facility in Grenoble and processed with DENZO and SCALEPACK (21) ( Table 1).
Structure Determination-The GlmS⅐Glc6P⅐DON and GlmS⅐Fru6P structures were solved by molecular replacement with AMoRe (22) using, respectively, the individual GlmS domains (Protein Data Bank codes 1GDO and 1MOQ) (8,9) or monomer A of the native protein (Protein Data Bank code 1JXA) (12) as model.
Refinement and Model Building-In the GlmS⅐Glc6P⅐DON structure, the two monomers in the asymmetric unit are related by a non-crystallographic 2-fold axis to form a compact dimer. In the GlmS⅐Fru6P structure, monomers B and C in the asymmetric unit form a dimer, whereas monomer A forms a dimer with its counterpart in the neighboring asymmetric unit. The refinement consisted of alternating rounds of manual fitting of the model to electron density maps using O (23) and isotropic refinement in CNS (24). The relatively high R-factors and large differences in the mean isotropic B-factors for the different domains of the Glms⅐Fru6P structure (Table 1) prompted us to use translation libration screw parameterization in REFMAC (25) to take into account translations and librations of pseudo-rigid bodies within the asymmetric unit of the crystal (26). Each synthase and glutaminase domain was treated as different TLS groups. Non-crystallographic symmetry restraints were used during the refinement of the GlmS⅐Fru6P structure in CNS and were released in REFMAC. Use of TLS parameters is reasonable as the GlmS⅐Fru6P structure is loosely packed with a solvent content of 60.8% and as the glutamine domain of chain C makes only one crystallographic contact with its symmetric molecule in the neighboring asymmetric unit. The analysis of the structures is based on monomer A, which is best defined in the electronic density in both structures. 99.7% of the residues in both structures are in the allowed regions of the Ramachandran plot. Residues in the disallowed regions include residues of the linker region between the two domains and Glu-91 (, 65.4°; , Ϫ116°), as previously observed (8,12), that is located at the edge between two ␤-sheets. Superposition of the structures have been made with SUPERPK. 3

RESULTS
The GlmS⅐Glc6P⅐DON structure is the first GlmS structure for which both sites contain a ligand, and this ternary complex represents the active conformation of the enzyme before the ammonia transfer step.
The comparison of this structure to the GlmS⅐Fru6P structure solved at 2.05 Å resolution, which is the state of the enzyme just before the glutamine binding step, therefore gives information about the conformational changes taking place upon glutamine binding.
Synthase Site-The refinement of the GlmS⅐Fru6P structure at 2.05 Å resolution using TLS parameters to account for the differences in mean isotropic B-factors between the different molecules in the asymmetric unit resulted in a structure of better quality compared with the previously determined structure at 3.1 Å resolution (12), as judged by the lower isotropic B-factors and the higher number of residues in the allowed region of the Ramachandran plot (Table 1). In addition, it allows visualization of water molecules that might play a significant role in catalysis (Fig. 3A) and assignment of the previously undefined nature of the sugar (Fig. 2A). Indeed, the F o Ϫ F c omit maps indicate that the bound sugar is in the linear form in both structures. The GlmS phosphoglucose isomerase activity may isomerize Fru6P to Glc6P when GlmS is crystallized in the presence of Fru6P and DON or Fru6P alone (7). The difference between the open-chain forms of Glc6P and Fru6P resides in the hybridization state of C1 and C2, which is difficult to determine at the current resolutions. However, the modeling of the linear forms of Glc6P or Fru6P and the examination of both the positive 3 P. Alzari, unpublished data. and negative contours of the difference density support the majority presence of Fru6P in the absence of DON (GlmS⅐Fru6P structure) or Glc6P in its presence (GlmS⅐Glc6P⅐DON structure). The overall structure of the synthase domain is very similar in the two structures, although some important differences are noticed at the synthase site (Fig. 2). First, the conformational change of Ser-401 is related to the different positions of the C2 oxygen of the sugar. Second, we observed conformational changes of some residues that constitute the walls of the channel: Lys503*, which participates to the channel of the neighboring subunit and the C-tail, in particular with the flipping of the peptide bond of Lys-603 and the conformational change of Ser-604 that are connected to the movements of Arg-26 and Trp-74 from the glutaminase domain. As discussed below, this opens the ammonia channel and activates the glutaminase function.
Glutaminase Site-In addition to the nucleophilic thiol group of Cys-1, the key catalytic elements for glutamine hydrolysis in GlmS are the ␣-amino group of Cys-1 and an oxyanion hole formed by N␦2 of Asn-98 and NH of Gly-99 in order to stabilize the tetrahedral interme-  The cavity is lined with residues that are conserved in the Ntn amidotransferases family: Cys-1, Arg-26, Arg-73, Asn-98, Gly-99, and Asp-123 as well as residues Thr-606, Thr-76, and Trp-74. A, Glms⅐Fru6P structure. The glutaminase site is empty, and the Q-loop is in an open conformation so that the glutamine pocket is exposed to the solvent. The carbonyl group of Arg-26 and water molecules hydrogen bind to the N-terminal amino group, whereas the carbonyl oxygen atom of Cys-1 hydrogen binds to a water molecule. The water molecules may mimic different positions of the water molecule that is supposed diate 6 (Fig. 1B). The electron density in the glutamine binding pocket of the GlmS⅐Glc6P⅐DON structure reveals that the thiol group of the nucleophilic cysteine residue is covalently modified by DON (Fig. 3B). In the GlmS⅐Fru6P structure, the thiol group of the nucleophilic Cys-1 is catalytically competent (Fig. 3A), although it does not point toward the active site in the N GlmS⅐Glu or N GlmS⅐Glu hydroxamate structures (8) (Fig. 3D). Therefore, it is likely that the active orientation of the thiol group of Cys-1 is triggered by acceptor binding. The glutaminase site of Ntn amidotransferase is composed not only by several conserved residues from the glutaminase site (3,27) but also by an oxygen atom of a residue of the synthase domain not conserved in this family that makes a direct hydrogen bond with the ␣-amino group of Cys-1 in the presence of glutamine. By making the corresponding hydrogen bond in GlmS through a conformational change upon DON binding (Fig. 3, B and C), Thr-606 enhances the nucleophilic character of the terminal ␣-amino group that acts as a general acid/base in glutamine hydrolysis (Fig. 1B) (5). In addition, the active conformation of Cys-1 is ensured by a hydrogen bond between its carbonyl oxygen atom and the guanidinium group of Arg-26 in the GlmS⅐Glc6P⅐DON structure (Fig. 3B), an interaction that is conserved in other Ntn amidotransferases where both sites are occupied (19,20).
The positioning of the side-chain NH 2 group of Asn-98 in order to form the oxyanion hole is another consequence of glutamine binding at the glutaminase site (Fig. 3, B and C). In the different structures of N GlmS (8) (Fig. 3D) and in the GlmS⅐Fru6P structure (Fig. 3A), the side-chain of Asn-98 does not adopt such a conformation. The proper structuring of the oxyanion hole requires occupation of both isomerase and glutaminase domains and is driven by a specific hydrogen bond with the guanidinium group of Arg-26, the conformational change of which is linked to the restructuring of loop 24 -29 (Fig. 3C). Thus, the conserved Arg-26-Gly-27 dipeptide previously defined as an "anchor" for both the backbone of Cys-1 and the side-chain of the asparagine involved in the oxyanion hole (3) is involved together with Thr-606 in the activation of the glutaminase function through specific hydrogen bonds.
In addition, comparison of the glutaminase sites of the GlmS⅐Glc6P⅐DON and GlmS⅐Fru6P structures shows that DON binding at the glutaminase site leads to a large conformational change of the Q-loop (Fig. 3, C and E). It had been previously anticipated that the glutamine loop would act as a gate keeper in controlling access to the glutaminase site in Ntn amidotransferases (28), but the present work is the first demonstration of the closure of the Q-loop upon DON binding to the acceptor-bound state of an amidotransferase. The most drastic structural change in the GlmS Q-loop concerns Trp-74, which adopts different conformations in the GlmS⅐Fru6P, GlmS⅐Glc6P⅐DON, and N GlmS⅐Glu structures (Fig. 3). These conformations are associated with different hydrogen binding patterns of its indole NH group as well as to a conformational change of Lys-503* resulting from steric hindrance (Fig. 2C). The 75°rotation of the indole ring of Trp-74 is crucial for ammonia transfer because the indole group blocks the channel in the GlmS⅐Fru6P structure but not in the GlmS⅐Glc6P⅐DON structure (see below). Trp-74 is therefore a major actor in coupling glutamine binding to ammonia transfer. In addition, the conformational change of the Q-loop upon DON binding allows a precise positioning of the glutamine analog in the active site. Indeed, it allows an ionic interaction between Arg-73 from the Q-loop and the ␣-carboxyl group of DON (Fig. 3B). Moreover, Asp-123, which belongs to loop 121-125 that is also involved in the closure of the glutaminase site, provides specific interactions with the ␣-amino group of the inhibitor (Fig. 3, B and E).
Interdomain Coupling-The C-tail is highly conserved among glucosamine-6P synthases (9). It is a fingerprint of the enzyme and has a fundamental role in the enzymatic function.
Role of the C-tail in Closing the Synthase Site-First, the C-tail makes contact between the glutaminase and the synthase domains and contributes to the sugar binding site through residues 603-605 (Fig. 4, A-C). The contact between the sugar and the C-tail is direct in the structures of C GlmS in complex with cyclic sugar (9), whereas it is mediated by numerous water molecules in the structures of native GlmS in complex with the open ring sugars (Fig. 4, A and B). Second, the C-tail shields the sugar binding site from bulk solvent when Fru6P is bound (Fig. 4C). Actually, in all the GlmS structures determined so far (9,10,12), the isomerase active site is occupied and in a closed conformation. The closure of the isomerase site is supposed to be triggered by Fru6P binding to the enzyme through a 20°rotation of the C-tail around the C␣-C bond of the conserved Pro-598 (Fig. 4C) (12).
Activation of the Glutaminase Function-The C-tail interacts with the glutaminase domain by providing binding pockets for Trp-74 and for the Tyr-28 hydroxyl group through hydrogen bonds with the backbone carbonyl of Pro-598 (Fig. 4C). In addition, the activation of the glutaminase function upon DON binding is ensured by residues of the C-tail: Thr-606, which provides direct hydrogen bonds to the ␣-amino to relay the acid/base catalysis of the terminal amino group (Fig. 2A). B, GlmS⅐Glc6P⅐DON structure. The Q-loop shields the glutaminase site from solvent. The ␣-carboxylic group of DON makes an ionic interaction with the guanidinium group of Arg-73 and hydrogen binds to NH and OH of Thr-76, NH of His-77, and N⑀2 of His-86 (omitted for clarity). The ␣-amino group of DON is hydrogen bonded to OH of Thr-76, the carboxylate group of Asp-123, and the carbonyl group of Gly-99. The carbonyl oxygen of DON hydrogen binds to N␦2 of Asn-98 and to NH of Gly-99. The carbonyl group of Cys-1 hydrogen binds to the guanidinium group of Arg-26 and to O␦1 of Asn-98. The orientation of the ␣-amino group of Cys-1 is maintained by a direct hydrogen bond with OH of Thr-606 in addition to the hydrogen bond with the carbonyl group of Arg-26 also observed in the GlmS⅐Fru6P structure. A water molecule located next to the cysteine ␣-amino group may represent one position of the solvent involved in the deacylation step of ␥-glutamyl-thioester 7 ( Fig. 2A). C, superposition of the glutaminase sites. The C␣s of the glutaminase domains (residues 1-72, 81-120, 125-136, 140 -200) were superimposed on each other (r.m.s.d., 0.59 Å). The water molecules are indicated as blue or orange spheres, respectively, for the GlmS⅐Fru6P or the GlmS⅐Glc6P⅐DON structures. Upon DON binding, the Q-loop closes the glutaminase site, the ␣-amino group of Cys-1 is activated through the direct hydrogen bond with OH of Thr-606 (r.m.s.d., 2.72 Å), and the oxyanion hole is formed by NH 2 of Asn-98 (r.m.s.d., 2.03 Å for all side-chain atoms). Conformational changes of loop 24 -29 (r.m.s.d., 0.95 Å) and the Q-loop (r.m.s.d. ϭ 1.43 Å), respectively, position Arg-26 to anchor the backbone of Cys-1 and the side-chain of Asn-98 and Arg-73 to provide the specificity for the ␣-carboxyl group of glutamine. The indole group of Trp-74 is oriented differently in the two structures (r.m.s.d. of 2.67 Å). Its NH group hydrogen binds to the guanidinium group of Arg-539*, which is involved in the enzyme dimerization (Fig. 5B), and the carbonyl group of Asn-600 either directly in the GlmS⅐Glc6P⅐DON or via a water molecule in the GlmS⅐Fru6P structure. D, superposition of the glutaminase sites of the N GlmS⅐Glu and GlmS⅐Glc6P⅐DON structures. The C␣s of each glutaminase domain (residues 1-22, 29 -49, 54 -58, 63-154, 158 -173, 177-200) were superimposed on each other (r.m.s.d., 0.34 Å). The hydrogen binding pattern is indicated only for the N GlmS⅐Glu structure (PDB code 1GDO, in gray) (8). The Q-loop is in a closed conformation, covering the glutaminase site, in both structures. In the N GlmS⅐Glu structure, Cys-1, Arg-26, and Asn-98 are not oriented in a way that supports catalysis. The thiol group of Cys-1 points away from the glutamine binding site (r.m.s.d., 2.55 Å). The presence of a charged carboxylate group in the product instead of the amide group of the substrate may trigger a reorientation of the cysteine residue so that an ionic interaction occurs between the N-terminal group and the Glu ␥-carboxylate group and so that repulsion takes place between the thiol group of Cys-1 and the Glu ␥-carboxylate group. In addition, the N-terminal group of Cys-1 hydrogen binds to the carbonyl group of Thr-72 (not shown for clarity) and to O␦1 of Asn-98, and the oxyanion hole is not formed. In the GlmS⅐Glc6P⅐DON structure, Asn-98 rotates by 103°(r.m.s.d., 3.06 Å for N␦2) to form, together with Gly-99, the oxyanion hole (Fig. 1A).   (12). D, the C-tail forms part of the ammonia channel. Binding of DON at the glutaminase site triggers the opening of the ammonia channel. The channel is formed mainly by main-chain atoms of the C-tail, indicated as a coil, and by the side-chains of residues Leu-601, Ala-602, Val-605, Thr-606, Val-607, and Trp-74, indicated as sticks. The accessible surface of the channel in the GlmS⅐DON⅐Glc6P structure calculated with PYMOL and a probe radius of 1.4 Å is represented as a mesh surface and is colored as a function of the atoms forming it (oxygen in red, carbon in orange, and nitrogen in blue). In this structure, the indole group of Trp-74 (in orange) lines the surface of the channel, which is open. The position of the indole group of Trp-74 that blocks the channel in the GlmS⅐Fru6P structure has been superimposed in blue. The 75°rotation of the indole of Trp-74 is measured by the differences in the C-C␣-C␤-C␥ dihedral angle. Water molecules in the channel form a hydrogen bond network with the backbone atoms of residues lining the channel as well as OH of Thr-606. These water molecules, which may reflect the position of ammonia in the channel, are likely absent during the ammonia transfer step.
group of Cys-1 and to Asn-98, and Lys-603 and Ser-604, which undergo conformational changes coupled to that of Arg-26, which is involved in the positioning of these two catalytic residues (Fig. 4B).
Opening of the Ammonia Channel-The x-ray structures of GlmS reveal that the glutaminase and synthase sites are separated by 18 Å. Residues 601-607 of the C-tail form part of an internal ammonia channel that connects the two active sites (Fig. 4D). In the GlmS⅐Fru6P structure, the channel is totally closed by the indole ring of Trp-74 (12), and we have shown that DON binding induces the closure of the Q-loop together with a 75°rotation of the Trp-74 indole side-chain (Fig. 3). The consequence of these conformational changes upon glutamine analog binding is the opening of the ammonia channel, allowing the connection between the two active sites (Fig. 4D).
Hinged Movement of the Glutaminase Domain Relative to the Synthase Domain-Another important change between the GlmS⅐Fru6P and GlmS⅐Glc6P⅐DON structures is a hinged movement of 21°between the glutaminase domains when the synthase domains are superimposed (Fig. 5A). The pivot point is near residue Asp-29 and the extremity of the Q-loop (Fig. 5B). Ala-75 and Thr-76 from the Q-loop participate in the dimer interface by interacting with the 524*-539* ␣-helix of the synthase domain of the neighboring monomer. Arg-539 is highly conserved in all known GlmS sequences, and the important salt bridge between Asp-29 and Arg-539* is conserved in the GlmS⅐Fru6P and GlmS⅐Glc6P⅐DON structures. Therefore, the hinged movement is necessary to allow the closure of the Q-loop without changing the dimer interface.

DISCUSSION
In GlmS, the coupling between the two active sites is especially efficient because ammonia cannot be used as a nitrogen donor. Hydrolysis of glutamine occurs significantly only when Fru6P is bound to the synthase site. Thus, the enzyme is supposed to adopt at least three different states (12): (i) The two active sites are empty, the C-tail and the Q-loop are in open conformations, the glutaminase function is inactive, and the ammonia channel is not formed. (ii) The binding of the sugar at the synthase site initiates an interdomain signal transduction that activates the glutaminase function. The C-tail closes the synthase site and orients the thiol group of Cys-1 in an active conformation. A transient channel is formed that is, however, blocked by the side-chain of Trp-74. (iii) Upon glutamine binding, the glutaminase site is fully activated and the ammonia channel is open. In this work, we have visualized the conformational changes that take place during this step by comparing the GlmS⅐Fru6P and the GlmS⅐Glc6P⅐DON crystal structures. These x-ray experiments provide the first structural evidence of glutaminase activation and channel opening upon glutamine binding in amidotransferases. Indeed, the changes observed in GlmS upon glutamine analog binding to the acceptor-bound state of the enzyme were not seen in ferredoxin-dependent glutamate synthase, which is the only other amidotransferase that has been crystallized in the presence of the acceptor alone and in the presence of both acceptor and glutamine analog, because of crystal packing (20). DON binding to GlmS induces the closure of the glutaminase site by a conformational change of the Q-loop accompanied by a 21°rotation of the glutaminase domain relative to the synthase domain, in order to maintain the dimer interface. These changes restrict the access of solvent to the glutaminase site, protect ammonia from protonation, and prevent it from escaping into the surrounding medium. The Q-loop is therefore a critical element in signaling between the two active sites. We have shown that glutamine analog binding to GlmS activates the glutaminase function by positioning Asn-98 to form the oxyanion hole and enhancing the nucleophilic character of the ␣-amino group of Cys-1. The communication between the two active sites is mediated by residues forming part of the channel, Arg-26 and the C-terminal residues including Thr-606, the only residue from the synthase domain participating to the glutamine binding site.