Severine I Gharbi

Marketa J Zvelebil

Stephen J Shuttleworth

Tim Hancox

Nahid Saghir

John F Timms

Michael D Waterfield

Professor M D Waterfield Frs

Exploring the specificity of the PI3K family inhibitor LY294002

Keywords: EAS, Epoxy-activated sepharose, CK2, casein kinase 2, GSK3, Glycogen synthase, Mass spectrometry, PIP3, Phosphatidylinositol

The Phosphatidylinositol 3-kinases (PI3Ks) regulate cellular signaling networks that are involved in processes linked to the survival, growth, proliferation, metabolism and specialized differentiated functions of cells. The subversion of this network is common in cancer and has also been linked to disorders of inflammation. The elucidation of PI3K physiological function has come from pharmacological studies, which use the enzyme inhibitors Wortmannin and LY294002, and from PI3K genetic knockout models of the effects of loss of PI3K function. Several reports have shown that LY294002 is not exclusively selective for the PI3Ks, and could in fact act on other lipid kinases and additional apparently unrelated proteins. Since this inhibitor still remains a drug of choice in numerous PI3K studies (over 500 in the last year), it is important to establish the precise specificity of this compound. We report here the use of a chemical proteomic strategy in which an analogue of LY294002, PI828, was immobilized onto epoxy-activated sepharose beads. This affinity material was then used as a bait to fish-out potential protein targets from cellular extracts. Proteins with high affinity for immobilized PI828 were separated by 1D-gel electrophoresis and identified by LC-MS/MS. This study reveals that LY294002 not only binds to class I PI3Ks and other PI3K-related kinases, but also to novel targets seemingly unrelated to the PI3K family.

Introduction

Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases responsible for the phosphorylation of phosphatidylinositols on the D3 position of their inositol ring. The evolution of this family of enzymes and its critical role in the life, death, growth, proliferation, metabolism and in differentiated cell function has been recently reviewed [START_REF] Engelman | The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism[END_REF]. The family is made up of 14 enzymes separable into four classes of which Class I, II & III are lipid kinases and class IV are related protein kinases [START_REF] Carpenter | Phosphoinositide kinases[END_REF][START_REF] Vanhaesebroeck | Signaling by distinct classes of phosphoinositide 3-kinases[END_REF]. The most studied are the class I PI3Ks, which are sub-divided into class IA and class IB. Following receptor activation, the class I PI3Ks are recruited to the membrane where they generate a pool of the lipid second messenger, phosphatidylinositol [START_REF] Vanhaesebroeck | Signaling by distinct classes of phosphoinositide 3-kinases[END_REF][START_REF] Pawson | Assembly of cell regulatory systems through protein interaction domains[END_REF][START_REF] Rameh | The role of phosphoinositide 3-kinase lipid products in cell function[END_REF] triphosphate (PIP3). The transient production of PIP3 initiates a cascade of events starting with the recruitment and activation of effector proteins which carry PIP3 binding domains (PH, FYVE, ENTH) [START_REF] Pawson | Assembly of cell regulatory systems through protein interaction domains[END_REF]. PDK1 and Akt, for example regulate a multitude of targets [START_REF] Rameh | The role of phosphoinositide 3-kinase lipid products in cell function[END_REF]. Due to the pivotal role of the PI3K/Akt axis in cellular physiology, its perturbation has been directly linked to various diseases, most notably cancer, immune disorders, inflammatory disease and diabetes [START_REF] Drees | Therapeutic potential of phosphoinositide 3-kinase inhibitors[END_REF]. Indeed, over-expression of PI3K [START_REF] Rogers | The phosphoinositide 3-kinase signalling pathway as a therapeutic target in squamous cell carcinoma of the head and neck[END_REF] with recent evidence of oncogenic and probably activating mutations of p110 [8,[START_REF] Samuels | High frequency of mutations of the PIK3CA gene in human cancers[END_REF] and loss of or reduced activity of the tumor suppressor PIP3 phosphatase PTEN [10] are reported during tumorigenesis. Many other genes in the PI3K pathway are also tumor suppressors which validates the importance of this pathway in cancer and the PI3K network as a target for cancer therapy [START_REF] Hennessy | Exploiting the PI3K/AKT pathway for cancer drug discovery[END_REF]12].

In order to develop drugs that modulate PIP3 levels it is crucial to delineate the specific biological functions of each isoform of the different classes of the PI3K family. Several approaches have been used to alter protein function and recent specific genetic animal models are uncovering isotype selectivity [START_REF] Brachmann | Role of phosphoinositide 3-kinase regulatory isoforms in development and actin rearrangement[END_REF][START_REF] Okkenhaug | Impaired B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice[END_REF][START_REF] Foukas | Critical role for the p110alpha phosphoinositide-3-OH kinase in growth and metabolic regulation[END_REF]. However, complementary chemical biology studies are required to help further elucidate specific class I PI3K isoform functions. Most of the small molecule PI3K inhibitors developed to date are ATPcompetitive inhibitors. The first described PI3K inhibitors which were considered as relatively specific were Wortmannin (Wm) [16,[START_REF] Yano | Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells[END_REF], a naturally occurring metabolite of Penicillium funiculosum, and LY294002 [START_REF] Vlahos | A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one (LY294002)[END_REF], that was derived from the flavonoid quercetin (Figure S1). Although these compounds are invaluable tools to study the PI3K pathway, they have a broad inhibitory profile across different classes of PI3Ks. Indeed, LY294002 was shown to inhibit mTOR and DNA-PK [START_REF] Brunn | Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002[END_REF] but also other protein kinases, such as casein kinase 2 (CK2) and Pim-1 [START_REF] Davies | Specificity and mechanism of action of some commonly used protein kinase inhibitors[END_REF][START_REF] Jacobs | PIM-1 ligand-bound structures reveal the mechanism of serine/threonine kinase inhibition by LY294002[END_REF]. Furthermore, other PI3K-independent effects directly attributed to LY294002 have been reported [22][START_REF] Choi | LY294002 inhibits monocyte chemoattractant protein-1 expression through a phosphatidylinositol 3-kinase-independent mechanism[END_REF][START_REF] Kim | LY294002 inhibits LPS-induced NO production through a inhibition of NF-kappaB activation: independent mechanism of phosphatidylinositol 3-kinase[END_REF][25][START_REF] Yamaguchi | Activating transcription factor 3 and early growth response 1 are the novel targets of LY294002 in a phosphatidylinositol 3-kinase-independent pathway[END_REF][START_REF] Tolloczko | LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] affects calcium signaling in airway smooth muscle cells independently of phosphoinositide 3kinase inhibition[END_REF]. For example, LY294002 was directly implicated in the inhibition of Ca 2+ signaling [START_REF] Tolloczko | LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] affects calcium signaling in airway smooth muscle cells independently of phosphoinositide 3kinase inhibition[END_REF] and transcription factors such as NF-B [START_REF] Kim | LY294002 inhibits LPS-induced NO production through a inhibition of NF-kappaB activation: independent mechanism of phosphatidylinositol 3-kinase[END_REF]. However, the direct targets of LY294002 involved in these processes have not been defined and we suspect that several targets may be involved. Currently, the validation of inhibitor specificity remains closely linked to previous knowledge of potential targets through modeling analysis or in vitro activity assays. Recent reports have revealed a new approach to assess drug specificity by direct immobilization of the small molecule inhibitors onto a solid phase and subsequent identification of bound proteins using optimized proteomic methods [28,[START_REF] Knockaert | Intracellular Targets of Paullones. Identification following affinity purification on immobilized inhibitor[END_REF]. We describe here the use of an LY294002-derived matrix to isolate and identify its direct molecular targets and to further understand reported off-target effects of this compound.

Materials and Methods

Reagents

Tissue culture media and fetal calf serum were from Invitrogen, Gibco, UK. Antibodies to p110 and p110 were a kind gift from Dr Bart Vanhaesebroeck (LICR, London, UK).

Antibodies to p85 and VCP were from Santa Cruz Biotechnology (CA, US). The antibody to ALDH was from BD Transduction Laboratories (CA, US). The antibody to mTOR was from Cell Signaling Technology (MA, US). LY294002 and LY303511 were from Merck Biosciences Ltd. (CA, US). Synthesis of 8-bromochromenone was carried out by Charnwood Molecular. PI828 and PI103 were synthesized by PIramed Ltd. All other reagents were from Sigma-Aldrich (MO, US) unless otherwise stated.

Synthesis of PI828

The precursor to LY294002 and PI828, 8-bromo-2-morpholin-4-yl-chromen-4-one was prepared as previously described [START_REF] Abbott | Synthetic Studies of the Phosphatidylinositol 3-Kinase Inhibitor LY294002 and Related Analogues[END_REF]. To prepare PI828 (Fig. 1A), a suspension of 161 mM 8-bromo-2-morpholin-4-yl-chromen-4-one, 177 mM 4-(4,4,5,5-tetramethyl- [START_REF] Abbott | PDE2 inhibition by the PI3 kinase inhibitor LY294002 and analogues[END_REF] dioxaborolan-2-yl)-phenylamine, 484 mM sodium carbonate in toluene/Ethanol (EtOH) was flushed with argon. 8 mM Dichlorobistriphenylphosphine palladium (II) was added and the mixture heated in a microwave reactor for 1h at 120°C. The crude mixture was partitioned between dichloromethane and water. The combined organic layers were washed with brine, separated and dried (MgSO 4 ). The crude product was evaporated onto silica and purified by flash column chromatography (5% 10% methanol:dichloromethane) to give a yellow solid which was further purified by trituration in ether:ethyl acetate to furnish the title compound as a pale yellow solid (177 mg , 49%).

Production of Immobilized PI828

Epoxy-activated Sepharose 6B (EAS) beads (GE Healthcare, 1 g) were incubated overnight at 55°C with 2 volumes of 20 mM PI828 in 50% (v/v) DMF/0.

Cell lysis and affinity purification experiments

HeLa cells (2.5x10 9 cells, Cilbiotech, Belgium) were lysed in 30ml of lysis buffer A ( 20mM Hepes (pH 7.5), 150 mM NaCl, 0.25% (v/v) Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM DTT supplemented with protease and phosphatase inhibitors). WEHI231 cells were grown in suspension in RPMI1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 50 µM -mercaptoethanol and antibiotics. For large-scale experiments, 15 mg of total cellular extract were adjusted to 1 M NaCl and incubated with 300 µl of 50% bead slurry for 4 h. For competition assays, lysates were pre-incubated with 1 mM of LY294002 or PI828 in lysis buffer A supplemented with 10 mM ATP, 20 mM MgCl 2 .

Affinity complexes were washed extensively in lysis buffer A adjusted to 1M NaCl and then in lysis buffer A. Complete elution was achieved in 1 x Laemmli sample buffer with boiling. Specific elutions were also carried out with free inhibitor (LY294002, PI828, PI103, LY303511) in lysis buffer A supplemented with 10 mM ATP, 20 mM MgCl 2 .

Protein separation and Mass spectrometry

Eluted proteins were separated by 1D gel electrophoresis and 1/10 th of the sample was kept for Western Blotting analysis. Preparative gels were stained with colloidal coomassie blue G-250 (Merck) and the whole lane dissected into smaller bands. An ingel digestion procedure was followed as previously described

Expression of recombinant VCP and binding assays

The wild type (WT) and ATP hydrolysis mutant (AA) p97/VCP constructs were a kind gift from Dr. Y Ye (NIDDK/NIH, MD, US) [33]. Full length mouse His-tagged p97/VCP was cloned into a pQE9 vector (Qiagen Ltd.) [33, 34] and expressed in E.coli following induction with 0.5 mM IPTG. Recombinant proteins were purified as described [34]. 2 g of WT and AA VCP were incubated with 10 l of beads slurry and pull-downs were carried out as described in the large-scale experiments.

Docking studies

The crystal structure of p110 [START_REF] Walker | Structural determinants of phosphoinositide 3kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine[END_REF][START_REF] Walker | Structural insights into phosphoinositide 3-kinase catalysis and signalling[END_REF] was used for homology modeling and inhibitor docking studies, although only the catalytic region of the protein was utilized (residues 725 to 1092). The models were constructed as described previously [START_REF] Pirola | Activation loop sequences confer substrate specificity to phosphoinositide 3-kinase alpha (PI3Kalpha). Functions of lipid kinase-deficient PI3Kalpha in signaling[END_REF]. Docking was carried out using the application GOLD (Cambridge Crystallographic Data Centre, Cambridge, UK), with GoldScore and point of origin set to C 5 of ATP from which a 10 Å pocket was defined. As a test of accuracy, the docked structure of PI828 was compared to the crystallographically determined structure [START_REF] Walker | Structural determinants of phosphoinositide 3kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine[END_REF]. The docked and structural coordinates and orientation of PI828 were in very good agreement. A further docking study of LY294009 and PI828 in both VCP (PDB number 1R7R) and ALDH2 (PDB number 1AG8) was carried out. For ALDH2, two pockets were identified, but the NAD-binding pocket proved to be the most probable site of interaction.

Results

Design and Immobilization of PI828; Validation of the Binding Assay

An analogue of LY294002 was designed following consideration of its binding mode to p110 as determined by X-ray crystallography [START_REF] Walker | Structural determinants of phosphoinositide 3kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine[END_REF]. The crystal structure indicated that the morpholine oxygen formed a hydrogen bond with the hinge region Val882, with a further putative interaction existing between the carbonyl oxygen and Lys833.

Significantly, the 4-position hydrogen of the exocyclic phenyl substituent, which occupies the ribose binding pocket, was seen to extend out towards solvent. Based on this structural information, we chose to incorporate an amine linker at this position to use as a site of attachment to the resin (Fig. 1A; synthesis and immobilization in supplemental data). The precursor to the immobilized ligand, PI828, showed encouraging biochemical inhibition of class I PI3Ks (Table S1). Indeed, PI828 was more potent than LY294002, particularly against p110 . From our modeling study, we postulated that this increase in potency may arise from hydrogen bonding of the PI828 amine protons with Glu858 and Asp862 in the catalytic site (Fig. 1B), residues which are unique among all the PI3K class I isoforms (Zvelebil et al, submitted manuscript). PI828 was immobilized onto epoxyactivated sepharose (EAS) beads using a procedure similar to that previously developed [28]. Binding of PI3Ks was first examined using recombinant p110 and then from WEHI231 cells and PI3K catalytic subunits were detected by western blotting (Fig. 1C).

Affinity binding conditions were mainly based on previously published work [28] and no binding to blocked EAS without immobilized inhibitor was observed (Fig. 2).

Identification of protein targets of PI828 and Evaluation of Relative Binding Affinity

We next used the PI828-matrix to perform large-scale pull-down assays in human epithelial cells (HeLa cells) (Table S2) and in the mouse lymphoma B-cell line WEHI231 (Table S3), which expresses high levels of p110 [START_REF] Bilancio | Key role of the p110delta isoform of PI3K in Bcell antigen and IL-4 receptor signaling: comparative analysis of genetic and pharmacologic interference with p110delta function in B cells[END_REF]. In the first instance, proteins were eluted in Laemmli sample buffer to ensure complete elution from affinity matrix. In later experiments, specific elution was carried out with LY294002 to evaluate binding profiles.

Reproducible protein identification from both cell types was observed although unique proteins were also found which may be related to different protein expression patterns of the two cell types (Fig 2A and Tables S2 andS3). PI3K catalytic subunits were highly represented in the bound fraction, and the most abundant isoform was p110 The p85 regulatory subunits were also present, which reflects the strong interaction between these two subunits. The class III PI3K, vps34, was also identified together with Beclin-1, a protein known to interact with vps34 [39]. No PI3K-related kinases were identified by mass spectrometry, which may be attributed to their low copy numbers and high molecular weights, although, mTOR was detected by immunoblotting (Fig. 2B). The type III phosphatidylinositol-4 kinase, (230 kDa isoform) was also identified, correlating with reports showing its sensitivity to both Wm and LY294002 [START_REF] Downing | Characterization of a soluble adrenal phosphatidylinositol 4-kinase reveals wortmannin sensitivity of type III phosphatidylinositol kinases[END_REF][START_REF] Meyers | Cloning and characterization of a wortmannin-sensitive human phosphatidylinositol 4-kinase[END_REF]. The PI828-matrix also bound to an array of proteins with various functions and substrate specificities.

These included proteins with a capacity for binding ATP, NAD(P)H, nucleotides and acetylated histones (Fig. 3A and 3B and see below).

Competitive binding with an excess of free inhibitor (1 mM LY294002 or PI828)

was carried out to validate binding specificities (Fig. 2A and2B). As well as the PI3K isoforms, other previously reported targets of LY294002, such as mTOR, CK2 and GSK3 , showed reduced binding in the presence of excess free inhibitor (Fig. 2B). On the other hand, bands corresponding to the family of dehydrogenases showed higher affinity for the PI828-matrix (Fig. 2A andB). Sequential competitive elution experiments with increasing concentrations of LY294002 (Fig. 4) demonstrated that the bound proteins had different binding strength for the affinity matrix. The ALDH2 interaction was strong and 10 mM of LY294002 or SDS were necessary to elute it from the PI828-matrix, suggesting high affinity to the beads (Fig. 4A and4B). We next used this affinity platform to assess binding profiles in competing concentrations of PI103 and LY303511 (Fig. 4).

PI103 is a potent inhibitor of p110 at low nanomolar concentration, as recently reported [START_REF] Fan | A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma[END_REF]43], and LY303515 is a structurally related analogue of LY294002 which has an amine substitution of its morpholine oxygen group, therefore altering its ability to inhibit PI3Ks [START_REF] Ding | Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils[END_REF]. As expected, LY303511 was less efficient at eluting p110 than LY294002 or PI103. PI103 was unable to efficiently elute mTOR in our competition assay although it can inhibit its activity [START_REF] Fan | A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma[END_REF] (Fig. 4). Both, CK2 and GSK3 were eluted at low concentrations of LY303511 and PI103 suggesting a high affinity for these inhibitors.

Evaluation of putative targets of LY294002

Some of the putative targets were further evaluated to address the functional impact of LY294002 on their activity. Casein kinase 2 (CK2), a previously reported target of LY294002 [START_REF] Davies | Specificity and mechanism of action of some commonly used protein kinase inhibitors[END_REF], showed robust binding to the PI828-beads with complete depletion of CK2 from samples achieved under the conditions used (data not shown). Notably, both catalytic and regulatory subunits of CK2 were recovered. The binding was also shown to be selective (Fig. 2B and4B) and CK2 was catalytically inhibited by LY294002 (Table 1). LY294002 was also able to reduce the kinase activity of both isoforms of the serine/threonine kinase Glycogen synthase kinase 3 (GSK3 and ) (Table S4). Another PI3K un-related target was the p97/Valosin-containing protein (VCP), a member of the type II AAA ATPase family. The presence of p97/VCP and other chaperones with ATPase activity in the PI828 pull-down assay is interesting since it suggests a novel class of targets of LY294002. To confirm our observation, we compared binding of recombinant wild type p97/VCP (WT), and a mutant defective in ATP binding (K251A; K524A, AA) to the PI828-matrix (Fig. 5A) [33]. This validated the direct interaction of VCP with the PI828-matrix, but also showed that these critical lysines are not required for binding to PI828, since similar binding was observed for the WT and mutant VCP.

We also characterized the binding by competing with an excess of free compound or ATP

showing that high concentration of free inhibitor (2 mM) plus ATP (10 mM) were required to reduce VCP binding (Fig. 5A). Homology modeling based on the crystal structure of ATP-bound VCP allowed both LY294002 and PI828 with its linker to be docked into the ATP binding site (Fig. 5B). In the LY294002-bound form, one putative hydrogen bond was observed with Ile380 but also between the linker and Arg256.

Furthermore, the linker was exposed to the solvent channel, providing enough space for the bulky matrix not to impede binding. Thus, it appears that VCP may be a direct target of LY294002 in cells, which may perturb its chaperone functions.

Discussion

The use of the chemical proteomic platform developed here has revealed a new subset of possible targets for LY294002 whose inhibition could affect various cellular processes particularly metabolism, transcription or protein trafficking and dynamics (Fig. 3). The major non-lipid kinases identified in this study have been previously reported (CK2, mTOR, GSK3 ), which emphasizes the thoroughness of this assay. It is likely that the PI3K-independent cellular effects observed with LY294002 are in fact due to inhibition of one or more of these three kinases. The serine/threonine kinase CK2 plays a putative role in proliferation and other cellular processes through activation of transcription factors and other cell signaling proteins and its overexpression in cancer has been reported [START_REF] Landesman-Bollag | Protein kinase CK2 in mammary gland tumorigenesis[END_REF]. One possible mechanism is that inhibition of CK2 by LY294002 could affect phospholipase C and calcium signaling [START_REF] Tolloczko | LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] affects calcium signaling in airway smooth muscle cells independently of phosphoinositide 3kinase inhibition[END_REF]. Additionally, GSK3 is involved in the regulation of glycogen metabolism through the inactivation of glycogen synthase, but it also has an important role in regulating many signaling pathways [START_REF] Doble | GSK-3: tricks of the trade for a multitasking kinase[END_REF]. This kinase is a known substrate of Akt [START_REF] Cross | Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B[END_REF] and is therefore regulated downstream of the class I PI3Ks.

It will therefore be important to note the direct effect of LY294002 and other PI3Kspecific inhibitors on this enzyme in future studies. Other targets had also previously been reported to be directly inhibited by LY294002, such as the cAMP phosphodiesterase 2 (PDE2) (Tables 2 and3 Supplemental data) [START_REF] Abbott | PDE2 inhibition by the PI3 kinase inhibitor LY294002 and analogues[END_REF]. However, the kinase Pim-1 was not identified in our assay and its binding to the PI828-matrix needs to be assessed by immunoblotting. Absence of Pim-1 could however reflect the proposed binding mode of LY294002 to this kinase where the orientation of LY294002 is distinct from p110 [START_REF] Jacobs | PIM-1 ligand-bound structures reveal the mechanism of serine/threonine kinase inhibition by LY294002[END_REF].

Under this conformation, it is possible that the bulky matrix disrupts the binding to our immobilized compound. We were also able to validate the direct binding of VCP to PI828-matrix in vitro and docking studies revealed a putative binding mode to the ATP binding pocket. It will be interesting to further evaluate the effect of LY294002 on the activity of this ATPase, since it seems to play a decisive role in the control of the ubiquitin-degradation machinery and protein trafficking [49]. Metabolite kinases were also found to be putative targets of LY294002, including fructosamine 3-kinase, ketosamine 3-kinase, galactokinase, phosphofructokinase and pyridoxal kinase (PXDK).

The latter is involved in the metabolism of vitamin B6 and was exclusively found in the HeLa pull-down experiments, which may reflect the reported low expression of PXDK in lymphomas [START_REF] Kang | Genomic organization, tissue distribution and deletion mutation of human pyridoxine 5'-phosphate oxidase[END_REF]. Interestingly, this protein was found to bind roscovitine, an inhibitor of cyclin-dependant kinases [START_REF] Bach | Roscovitine targets, protein kinases and pyridoxal kinase[END_REF]. The potential effect of LY294002 on PXDK activity requires further assessment. The most abundant protein bound to the PI828-matrix was aldehyde dehydrogenase 2 (ALDH2), one of five dehydrogenases identified. Previous chemical proteomic approaches also reported significant binding of dehydrogenase enzymes to inhibitor matrices [28,[START_REF] Knockaert | Intracellular Targets of Paullones. Identification following affinity purification on immobilized inhibitor[END_REF]. For example, Knockaert et al showed binding of paullone cyclin-dependant kinase inhibitors to malate dehydrogenase with reduction of enzymatic activity. We carried out homology modeling based on the crystal structure of ALDH bound to NADH, and in the most probable conformation, LY294002 was docked in the NAD-binding pocket, suggesting direct competition with substrate binding (data not shown). A putative hydrogen bond between the backbone of the enzyme and the linker region of PI828 could explain the strong interaction observed in the competition experiments. Another group of identified proteins contained bromodomains (Tables S2 andS3), suggesting that LY294002 (or PI828) may directly target these domains.

Bromodomain-containing proteins are a class of proteins capable of interacting with chromatin through acetylated histones [START_REF] Dey | The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis[END_REF] and which can act as transcriptional activators [START_REF] Farina | Bromodomain protein Brd4 binds to GTPase-activating SPA-1, modulating its activity and subcellular localization[END_REF][START_REF] Sinha | Bromodomain analysis of Brd2dependent transcriptional activation of cyclin A1[END_REF]. The major Brd protein present in our pull-down was Brd4. This represents an interesting target since it may correlate with a reported inhibitory effect of LY294002 on the regulation of transcription [START_REF] Kim | LY294002 inhibits LPS-induced NO production through a inhibition of NF-kappaB activation: independent mechanism of phosphatidylinositol 3-kinase[END_REF].

The goal of this work was the identification of novel targets of LY294002 to further understand its reported off-target effects. Our strategy has enabled the identification of novel targets un-related to PI3Ks whose inhibition could contribute to the cellular effects observed upon treatment with LY294002. This approach represents a valid tool to establish a global interaction map of compounds of interest. It will be interesting to further evaluate the effect of LY294002 on the activity of some of these proteins, which will provide useful information not only in the context of LY294002, but also for the validation of novel generations of more potent and specific inhibitors.

Furthermore, this approach has the potential to be used as a robust high-throughput platform to validate the specificity of new PI3K inhibitors and novel therapeutic agents. Binding assays with recombinant p110 -GST fusion protein or from a total cellular extract of WEHI 231 cells. Pull-downs in 10 l of beads slurry were carried out in high salt with increased concentration of recombinant enzyme or total extract. Levels of p110 and p110 were evaluated by western blotting with specific antibodies.

  [32]. Each sample (1/2 of total peptide mixture) was analyzed by LC-ESI-MS/MS using an Ultimate nano HPLC (Dionex Ltd, UK) with a PepMap C18 75-m inner diameter column (Dionex) at a flow rate of 300 nL/min, linked on line to a QTof-I mass spectrometer (Waters, UK).Processed mass spectra (MassLynx, Waters) were searched against the updated IPI human (HeLa) or mouse (WEHI 231) databases using Mascot version 2.0.02 (Matrix Sciences, UK). Identifications were accepted when a minimum of 2 individual peptide masses matched a protein (mass error of ±100 mmu allowing 1 missed cleavage), MOWSE scores were higher than the threshold value (P = 0.05), and the predicted protein mass agreed with the gel-based mass.

Figure 1 :

 1 Figure 1: Synthesis of the LY294002 derivative and evaluation of binding to class I PI3Ks. (A) Synthesis of PI828 (4) and the immobilized derivative, PI828-matrix[START_REF] Rameh | The role of phosphoinositide 3-kinase lipid products in cell function[END_REF] starting from the 8-Bromochromenone derivative (3). (B) Putative binding mode of PI828 in p110 as assessed by Molecular Modelling (using GOLD). Green-dotted lines represent putative hydrogen bonds. Residues D862 and E858 are specific for p110 . (C) Efficient binding to the PI828-matrix from recombinant enzyme and total cellular extract. Binding assays with recombinant p110 -GST fusion protein or from a total cellular extract of WEHI 231 cells. Pull-downs in 10 l of beads slurry were carried out in high

Figure 2 :

 2 Figure2: Identification of cellular targets of LY294002. A total cellular extract from WEHI231 cells grown in 10% FCS (15 mg) was incubated in batch with the PI828matrix or the control saturated EAS devoid of the compound. Specific binding was validated by competition assay with 1 mM of LY294002 or PI828. (A) Bound material was eluted in 2x Laemmli sample buffer separated on a 1D-PAGE and proteins were detected by colloidal coomassie staining. The entire lanes were dissected and proteins identified by LC-MS/MS. Identified proteins are reported on the gel image (B) In parallel, samples were analyzed by western blotting with specific antibodies to p110 , p110 , mTOR, ALDH1, Brd4, GSK3 and CK2 .

Figure 3 :Figure 4 :

 34 Figure 3: (A) LY294002-interacting proteins and their cellular functions. (B) Identified proteins share different substrate specificity

Figure 5 :

 5 Figure 5: The AAA ATPases p97/VCP is able to bind directly PI828-matrix. (A) Invitro binding experiment was carried out with recombinant VCP (WT: wild type; AA: mutant defective in ATP hydrolysis). Binding was carried out in various concentrations of inhibitor and ATP and bound proteins were eluted in 2x Laemmli sample buffer. Proteins were visualized by western blotting with anti-VCP antibodies. (B) PI828 was docked in the ATP binding pocket of VCP.

Table 1 : LY294002 is a potent inhibitor of CK2. In

 1

	-vitro lipid kinase assays were

Table 1 :

 1 LY294002 is a potent inhibitor of CK2. In-vitro lipid kinase assays were carried out with both compounds (LY294002 and PI828) for casein kinase 2 (CK2). Inhibitor concentrations for 50% inhibition (IC 50 ) were established with recombinant CK2.

	Corporate ID	Compound	p110	CK2(h) CK2 2(h) cSRC(h) Yes(h)
					Mean IC50 ( M)
	PI000828	PI828	0.173	0.149	1.127	>10,000 >10,000

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 15 Feb 2007 as manuscript BJ20061489 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

The authors would like to thank Henrik Daub and Klaus Godl for important advice and support in the immobilization of PI828. We are grateful of Yihong Ye for the VCP constructs and Bart Vanhaesebroeck for p110 antibodies. We would also like to thank Barbara Geering, Jaime Millán Martinez and Pedro Cutillas for constructive discussions. This work was funded by the Ludwig Institute for Cancer Research (SG, MW, MZ, JT) and Piramed Ltd (SS, TH, NS).


Jaros Aw Olchowy

Iwona Gabriel

S Awomir Milewski
email: milewski@chem.pg.gda.pl

S Awomir Milewski

Functional domains and interdomain communication in Candida albicans glucosamine-6-phosphate synthase

Keywords: domain, amidotransferase, glucosamine-6-phosphate synthase, sitedirected mutagenesis, intramolecular channel

Functional and structural properties of several truncated or mutated variants of Candida albicans glucosamine-6-phosphate synthase (Gfa1p) were compared to those of the wild-type enzyme. Fragments encompassing residues 1-345 and 346-712 of Gfa1p, expressed heterogenously in bacterial host as oligoHis fusions, were identified as functional domains: glutamine amide-hydrolysing (GAH) and hexose phosphate-isomerising (ISOM), respectively. It was found that native GAH is monomeric, while native ISOM forms tetramers, as does the whole enzyme.

Spectrofluorimetric and kinetic studies of the isolated domains, the 218-283Gfa1p mutein and the whole enzyme revealed that the binding site for the feedback inhibitor, UDP-GlcNAc, is located in the ISOM domain. Inhibitor binding affects amidohydrolysing activity of GAH and, as a consequence, the GlcN-6-P-synthetic activity of the whole enzyme. The 218-283 fragment is neither involved in ligand binding nor in protein oligomerisation. Comparison of the catalytic activities of V711FGfa1p, 709-712Gfa1p, W97FGfa1p and W97GGfa1p to those of the native Gfa1p and the isolated domains provided evidence for an intramolecular channel connecting the GAH and ISOM domains of Gfa1p. The channel becomes leaky upon deletion of amino acids 709-712 and in W97F and W97G mutants. The W97 residue was found to function as a molecular gate, opening and closing the channel. The W97G and V711F substitutions resulted in an almost complete elimination of the GlcN-6-P-synthetic activity with the retention of the amidohydrolase and sugar phosphate-isomerising activities.

INTRODUCTION

Glucosamine-6-phosphate /GlcN-6-P/ synthase, systemic name L-Glutamine:D-fructose-6-phosphate amidotransferase, EC 2.6.1. [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF], is an ubiquitous enzyme, catalysing the first committed step in the amino sugar biosynthetic pathway in prokaryotic and eukaryotic organisms. The enzyme catalyses a complex reaction: L-Gln + D-fructose-6-phosphate D-glucosamine-6-phosphate + L-Glu, that involves ammonia transfer and sugar phosphate isomerisation. GlcN-6-P synthase is an important point of metabolic control of amino sugar biosynthesis, the mammalian enzyme is proposed to be involved in the phenomenon of hexosamineinduced insulin resistance in diabetes [START_REF] Hebert | Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance[END_REF] and the fungal GlcN-6-P synthase has been proposed as a potential target in antifungal chemotherapy [START_REF] Borowski | Novel approaches in the rational design of antifungal agents of low toxicity[END_REF]. For all these reasons, the enzyme, especially its eukaryotic type, is an interesting subject of study. The prokaryotic GlcN-6-P synthase has been relatively well characterised. The Escherichia coli enzyme has been purified to homogeneity [START_REF] Badet | Glucosamine synthetase from Escherichia coli: purification, properties and glutamine-utilizing site location[END_REF], details of the molecular mechanism of catalysis have been deduced [4 and references cited therein] and its 3D structure has been determined [START_REF] Teplyakov | Channeling of ammonia in glucosamine-6-phosphate synthase[END_REF]. Previous studies on E. coli

GlcN-6-P synthase revealed that the enzyme is a homodimer. Each subunit is composed of two domains that can be released from the native protein upon limited proteolysis with chymotrypsin: the N-terminal glutamine amidohydrolase (GAH, residues 1-239) and the C-terminal ketose/aldose isomerase (ISOM, residues 240-608) [START_REF] Teplyakov | Channeling of ammonia in glucosamine-6-phosphate synthase[END_REF]. The former binds L-Gln and hydrolyses it to glutamate and ammonia. The latter utilises the released ammonia for the conversion of D-fructose-6-phosphate to D-glucosamine-6-phosphate. Such a modular structure is characteristic for all amidotransferases composed of an amidohydrolase domain fused with an acceptor domain [START_REF] Massière | The mechanism of glutaminedependent amidotransferases[END_REF]. It is assumed that ammonia generated at the amidohydrolase domain is transferred to the second domain through the intramolecular channel. In most amidotransferases the channel is accessible for an exogenous ammonia, however the GlcN-6-P synthase seems to be unique in its apparent inability to use this substrate [START_REF] Massière | The mechanism of glutaminedependent amidotransferases[END_REF]. The presence of a solvent-inaccessible, relatively long channel composed of several hydrophobic amino acid residues, was demonstrated in the crystal structure of E. coli GlcN-6-P synthase [START_REF] Teplyakov | Channeling of ammonia in glucosamine-6-phosphate synthase[END_REF]. However, some evidence was presented for the close proximity of the domains' active sites [START_REF] Leriche | Affinity labeling of Escherichia coli glucosamine-6-phosphate synthase with a fructose 6-phosphate analog. Evidence for proximity between the N-terminal cysteine and the fructose-6-phosphate-binding site[END_REF] and, recently, for a hinged movement of the amidohydrolase domain towards the isomerase domain, induced upon substrate binding [START_REF] Mouilleron | Glutamine binding opens the ammonia channel and activates glucosamine-6P synthase[END_REF].

Only limited structural data are available for the eukaryotic GlcN-6-P synthase.

There is little doubt that the essential active site amino acid residues are highly conserved among GlcN-6-P synthases. However, the eukaryotic enzyme differs from its prokaryotic counterpart in a number of features. The most important of these is the quaternary structure and regulation of the enzymatic activity. The eukaryotic GlcN-6-P synthase is undoubtedly homotetrameric, its activity is regulated by a feedback inhibition by UDP-GlcNAc, as well as by phosphorylation/dephosphorylation mediated by protein kinase A and protein phosphatase [START_REF] Milewski | Glucosamine-6-phosphate synthase -the multifacets enzyme[END_REF]. Moreover, the results of multiple sequence alignment analysis of the enzyme revealed the presence of a 30 -80 aa fragment of unknown function at the C-terminal part of the eukaryotic GAH. No such fragment was observed in any known prokaryotic versions of the enzyme [START_REF] Milewski | Glucosamine-6-phosphate synthase -the multifacets enzyme[END_REF].

In this work we describe the results of our studies on GlcN-6-P synthase from the human pathogenic fungus Candida albicans (Gfa1p), involving structural and functional characterization of several truncated or mutated variants of this enzyme.

MATERIALS AND METHODS

Bacterial strains and media

The Escherichia coli XL1-Blue strain /recA1, endA1, gyrA96, thi-

Plasmids, enzymes, and other materials

The plasmids used were: pET23b (Novagen), pUC19 (Fermentas), pUC19-CaGFA1 and pET23b-CaGFA1 [START_REF] Sachadyn | Purification to homogenity of Candida albicans glucosamine-6-phosphate synthase overexpressed in Escherichia coli[END_REF]. Restriction and modification enzymes were purchased from Fermentas, Lithuania and New England Biolabs, USA. Purification of His-tagged proteins was performed on Ni 2+ -IDA agarose (his Bind Resin, Novagen).

DNA molecular weight markers were from DNA-Gda sk II s.c., Poland. The GelCode Phosphoprotein Staining Kit was from Pierce, USA. Yeast Glc-6-P dehydrogenase and other reagents were from Sigma, USA.

DNA manipulations

Isolation of plasmid DNA was carried out according to the protocol of the Plasmid Mini kit (A&A Biotechnology, Poland). DNA fragments were isolated from agarose gels following the standard procedure of the DNA Gel-Out kit (A&A Biotechnology, Poland). DNA purification after enzyme treatment was performed according to the instructions in the DNA Clean-up kit (A&A Biotechnology, Poland).

DNA digestion with restriction enzymes was carried out according to the enzyme supplier's instructions. DNA fragments were ligated and Escherichia coli cell were transformed according to the standard methods [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF].

Construction of expression plasmids containing domain-coding sequences

The fragment of the GFA1 gene encoding residues 1-345 of Gfa1p was amplified from the pUC19-CaGFA1 plasmid by PCR. The primers used in the amplification were: GLN1-KN (Table 1) and GLN2-HX, 5'-cct gcc aag ctt ctc gag aat

Construction of expression plasmids containing truncated versions of GFA1

218-283GFA1. Two fragments were amplified from the pUC19-CaGFA1 template by PCR using the following primers: fragment 1 GLN1-KN (Table 1) and Ingln-X, 5'tcc agg ctc gag atc agt ttt aac acc aac caa taa tg-3', fragment 2 Infru-X , 5'-cct acc ctc gag ttc ttt tta tct tct gat cct gc-3' and Fru2-Eco (see above template by PCR using the following primers: Trpgly-5 (Table 1), GFA709X, 5'-atg acg ctc gag tta tga ttt agc caa gtt acg agg-3'. The PCR product was purified and digested with Bsp119I and XhoI to give 3 products, of which the 1052 bp one was isolated. The pET23b-CaGFA plasmid was digested with the same restriction enzymes and a 4666 bp fragment was isolated from the digest. The isolated fragments were ligated, and E. coli XL1-Blue cells were transformed with the ligation product. The resulting plasmid construct pET23b-CaGFA1 709-712 was isolated and its identity was confirmed by sequencing.

Construction of expression plasmids containing site-directed mutant versions of GFA1

The site-directed mutants W97G and W97F of GFA1 were constructed using the overlap extension method with pUC19-CaGFA1 as a template. Primers used for PCR are listed in Table 1. The V711F mutant was amplified from pUC19-CaGFA1 with Gln1-KN and Valphe primers. The PCR products were isolated, digested with MluI, KpnI and BglII (W97F), KpnI, KasI and BglII (W97G), Bsp119I and XhoI (V711F) and ligated to the pET23b-CaGFA1 vector, digested with the appropriate restriction enzymes. E. coli XL1-Blue cells were transformed with the ligation products. The resulting plasmid constructs pET23b-CaGFA1W97G, pET23b-CaGFA1W97F and pET23b-CaGFA1V711F were isolated and their identity was confirmed by sequencing. Construction of the pET23b-CaGFA1S208A plasmid was described previously [START_REF] Gabriel | Phosphorylation of glucosamine-6-phosphate synthase is important but not essential for germination and mycelial growth of Candida albicans[END_REF].

Protein expression and purification

Escherichia coli BL21(DE3)pLysS cells, transformed with the appropriate expression plasmid, were grown in LB medium at 37 C. Expression was induced by the addition of IPTG, 0.5 mM, to the cultures growing to OD 600 = 0.6 -0.8 and incubation was continued for another 5 h. Cells were harvested by centrifugation (5000 g, 10 min, 4 C).

The His-tagged putative domains of Gfa1p were purified by metal affinity chromatography. The bacterial pellet was suspended in buffer A (20 mM Tris-HCl pH 7.9, 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100, 0.5 mM PMSF) and the cells were disrupted by sonication on ice. The total lysate was centrifuged for 20 min at 16000 g, 4 C. The supernatant (crude extract) was applied to a Ni 2+ -IDA agarose (his Bind Resin, Novagen) column, bed volume 10 ml, equilibrated with four volumes of buffer A. Next, the column was washed with 20 ml of the same buffer, followed by washing with 40 mL of buffer W (20 mM Tris-HCl pH 7.9, 500 mM NaCl, 80 mM imidazole, 0.1% Triton X-100, 0.5 mM PMSF). The His-tagged proteins were eluted with two 5 ml portions of elution buffer E (20 mM Tris pH 7.9, 500 mM imidazole, 0.5 M NaCl, 0.1% Triton X-100, 0.5 mM PMSF). For further assays the eluates were concentrated by ultrafiltration using Vivaspin concentrators, 10 kDa cut-off limit (Viva Science Ltd, UK) at 7000 g and the buffer was exchanged for buffer B (25 mM potassium-phosphate, pH 7.5, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF) using the HiTrap Desalting Columns (Pharmacia LKB).

The wild-type Gfa1p, its truncated and site-directed mutant variants were purified by a multi-step procedure, essentially as described previously [START_REF] Sachadyn | Purification to homogenity of Candida albicans glucosamine-6-phosphate synthase overexpressed in Escherichia coli[END_REF].

Determination of enzymatic activities and enzyme kinetic analysis

The amidohydrolysing activity was determined using L--glutamyl-p-nitroanilide (GLUPA) as a substrate. Mixtures containing 1 mM GLUPA, 1 mM EDTA, 1 mM DTT and appropriately diluted enzyme preparation in 20 mM Hepes buffer, pH 7.5, were incubated at 25 C. In some experiments, Fru-6-P (D-fructose-6-phosphate), 10 mM was also included as a component of the incubation mixture. The released pnitroaniline was quantified spectrophotometrically at 420 nm, assuming 420 (pnitroaniline) = 9 300 M -1 cm -1 . One unit of specific activity was defined as an amount of the enzyme that catalysed formation of 1 mol of p-nitroaniline/min/mg protein.

The rate of L-glutamine hydrolysis was determined by quantification of the released ammonia with the Nessler's reagent. Mixtures containing 10 mM L-Gln, 1 mM EDTA, 1 mM DTT and an appropriately diluted enzyme preparation in 20 mM Hepes buffer, pH 7.5, were incubated at 25 C. Aliquots of 0.1 ml were withdrawn at 5 min intervals, diluted with 1.4 ml of water and 0.2 ml of a commercial solution of the Nessler's reagent was added. The mixtures were left for 10 min at room temperature and absorbance at 480 nm was measured. The released ammonia was quantified, assuming a molecular absorbance index for the yellow product of the Nessler reaction with a standard NH 4 Cl solution, 480 = 3 10 8 M -1 cm -1 .

The hexosephosphate-isomerising activity was determined using the Glc-6-P dehydrogenase-coupled assay, as described by Noltmann et al. [START_REF] Noltman | Isolation of crystalline phosphoglucose isomerase from rabbit muscle[END_REF]. Incubation mixtures containing 2 mM Fru-6-P, 0.5 mM NADP + , 1 mM EDTA, Glc-6-P dehydrogenase, 2.6 unit/ml and appropriately diluted GlcN . One unit of specific activity was defined as an amount of the enzyme that catalysed formation of 1 mol of Glc-6-P/ min/mg protein.

GlcN-6-P-synthetic activity was assayed as described previously [START_REF] Milewski | Oligomeric structure and regulation of Candida albicans glucosamine-6-phosphate synthase[END_REF].

For the determination of kinetic constants, the amidohydrolysing, Fru-6-Pisomerising or GlcN-6-P-synthetic activities were determined at variable (0 -10 mM) initial concentrations of appropriate substrates. The data were plotted as Michaelis-Menten graphs. The kinetic data were subjected to the nonlinear regression analysis using the Enzyme Kinetics Module 1.10 for Sigma Plot software. The K m and k cat values were determined in triplicate, to give the mean values S.D.

Phosphorylation of Gfa1p, Gfa1S208Ap and GAHp-His 6 in vitro

Gfa1p, Gfa1S208Ap or GAHp-His 6 (100 g/ml) were incubated for 30 min at 25 C with 10 M cAMP, 1 mM ATP, 10 mM EDTA, 40 mM NaF and, if necessary, with the specific kinase A inhibitor H-89, 10 g/ml. The reaction was initiated by the addition of the cAMP-dependent kinase from beef heart (30 units/ml). After 30 min, H-89 (10 g/ml) was added to each sample to stop the kinase A activity. Samples were subjected to SDS-PAGE analysis and assayed for amidohydrolysing or GlcN-6-P-synthetic activity.

Detection of phosphorylated proteins in SDS-PAGE gels

The gels obtained after SDS-PAGE separation of protein mixtures incubated with kinase A, were stained with the GelCode Phosphoprotein Staining Kit, according to the protocol provided by the manufacturer. Gel images were documented, the gels were destained by several washings with demineralised water and stained with Coomassie Brilliant Blue R-250 to visualise all the protein bands.

Steady state fluorescence measurements

Fluorescence emission spectra were obtained using the Perkin Elmer LS55 spectrofluorimeter at room temperature. The excitation wavelength was set at 300 nm for selective excitation of tryptophan fluorescence. Incubation mixtures contained 0.3 M of the respective domain, mutant or wild-type Gfa1p, 1 mM EDTA and/or a ligand at appropriate concentration in 25 mM phosphate buffer, pH 7.5. The incubation solution was stirred continuously in a 1.0 cm 1.0 cm cuvette. The bandwidths for both the excitation and the emission monochromators were 4 nm. The spectra were corrected for background and Raman scattering by subtracting buffer spectra containing all ingredients except the protein.

Molecular mass determination of a native protein

Size-exclusion chromatography was performed on Superdex 200 HR 10/30 (Pharmacia LKB), eluted at 0.5 ml/min with 25 mM potassium-phosphate buffer, pH 7.0, containing 0.15 M NaCl, 1 mM EDTA and 1 mM DTT. Protein elution was monitored at 280 nm. The molecular mass standards were: thyroglobulin (669 kDa), apoferritin (443 kDa), -amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa).

Other methods

The wild-type C. albicans GlcN-6-P synthase (Gfa1p) was overproduced in E. coli and purified to near homogeneity according to the multi-step procedure described by Sachadyn et al. [START_REF] Sachadyn | Purification to homogenity of Candida albicans glucosamine-6-phosphate synthase overexpressed in Escherichia coli[END_REF]. Construction and purification of His 6-Gfa1p and Gfa1p-His 6 was described previously [START_REF] Olchowy | Construction, purification and functional characterisation of His-tagged Candida albicans glucosamine-6-phosphate synthase expressed in Escherichia coli[END_REF]. Protein was determined by the Bradford method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. Discontinuous SDS-PAGE was performed by the method of Laemmli [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] with 10% separating gel and 5% stacking gel.

RESULTS

Structural alignment of bacterial and fungal Gfa1p sequences

Construction, expression, purification and characterization of the His 6 -tagged domains of C. albicans Gfa1p

The two-domain structure of E. coli GlcN-6-P synthase is well documented, as well as a role played by each domain in the mechanism of catalysis. As shown in Fig. 2, the GAH domain (residues 1-239) is connected with the ISOM (residues 249-608) by a flexible nonapeptide linker. In the first step of our present studies we constructed two separate fragments of the C. albicans Gfa1p and characterised their functional properties. One of them comprised residues 1-345 and another one -residues 346-712. Both constructs contained oligoHis tags to facilitate their purification, however in the 1-345 construct, the tag was located at the C-terminus, while in the 356-712 construct it was at the N-terminus. Such localisations were an obvious consequence of the results of our previous studies on the construction and functional properties of the N-and C-His-tagged versions of the complete enzyme. We showed that the presence of an oligoHis tag at either end almost entirely inactivated the GlcN-6-Psynthesising activity of the enzyme. On the other hand, the N-tagged enzyme lost the ability to hydrolyse L-Gln but not to interconvert Glc-6-P and Fru-6-P, while the opposite was found for the C-tagged enzyme [START_REF] Olchowy | Construction, purification and functional characterisation of His-tagged Candida albicans glucosamine-6-phosphate synthase expressed in Escherichia coli[END_REF].

Expression plasmids pET23b-GLN and pET23b-FRU were constructed and their identities were confirmed by restriction analysis and gene sequencing. The fusion proteins, called GAHp-His 6 and His 6 -ISOMp, respectively, were overproduced in E. coli BL21(DE3)pLysS cells that had been transformed with the respective overexpression plasmid. Densitometric analysis revealed that in each case the recombinant protein constituted 15-20% of the total cytoplasmic protein pool. GAHp-His 6 and His 6 -ISOMp were purified by metal-affinity chromatography using the Ni 2+ IDA-agarose resin. A single-step elution resulted in both cases in almost homogenous protein preparations ( 98% homogeneity), isolated with 79 -81% yield.

The exemplary results of the SDS-PAGE analysis of His 6 -ISOMp purification are shown in Fig. 3.

The purified GAHp-His 6 and His 6 -ISOMp were characterised in terms of their enzymatic activities and molecular mass/size and these properties were compared to those of the wild-type complete enzyme and its oligo-His-tagged fusions. The amidohydrolysing activity was tested using L--glutamyl-p-nitroanilide (GLUPA) as the substrate. The hexosephosphate-isomerising activity was determined with Fru-6-P as the substrate, by continuous monitoring of the Glc-6-P formation in the Glc-6-P dehydrogenase-coupled assay. The GlcN-6-P formation was followed by the modified Elson-Morgan reaction [START_REF] Milewski | Oligomeric structure and regulation of Candida albicans glucosamine-6-phosphate synthase[END_REF]. The obtained results are summarised in Table 2. As expected, both GAHp-His 6 and His 6 -ISOMp were completely devoid of the GlcN-6-P-synthetic activity. On the other hand, both fragments exhibited partial activities: GAHp-His 6 catalysed GLUPA hydrolysis and His 6 -ISOMp was able to isomerise Fru-6-P to Glc-6-P. Kinetic parameters of both reactions were very similar to those found for the wild-type intact enzyme. Catalytic properties of GAHp-His 6 were also very similar to those of Gfa1p-His 6 , while His 6 -ISOMp demonstrated catalytic properties almost identical to those of His 6 -Gfa1p. Therefore, there is little doubt that in C. albicans GlcN-6-P synthase, the 1-345 fragment constitutes the GAH domain and the remaining part is the ISOM domain.

Molecular mass of the native GAHp-His 6 , determined under non-denaturing conditions (size-exclusion chromatography), was 42 2 kDa and that of the denatured form of this protein (determined by SDS-PAGE) was 41 0.5 kDa. The values found for His 6 -ISOMp were: 159 2 kDa and 39.5 0.5 kDa, respectively.

One may therefore conclude that GAHp-His 6 is monomeric in solution, while His 6 -ISOMp is homotetrameric.

Our previous studies showed that C. albicans Gfa1p can be phosphorylated by a cAMP-dependent protein kinase and that the GlcN-6-P-synthetic activity of the enzyme increases upon protein kinase A-driven phosphorylation [START_REF] Milewski | Oligomeric structure and regulation of Candida albicans glucosamine-6-phosphate synthase[END_REF]. Moreover, we identified the S208 residue as an unique phosphorylation site [START_REF] Gabriel | Phosphorylation of glucosamine-6-phosphate synthase is important but not essential for germination and mycelial growth of Candida albicans[END_REF]. In the present studies we checked whether the isolated GAH domain of Gfa1p, containing serine 208, was a substrate for a commercially available cAMP-dependent protein kinase from beef heart. GAHp-His 6 was incubated with the protein kinase under conditions optimal for protein phosphorylation and its amidohydrolysing activity was monitored.

For comparison, the same activity was measured for Gfa1p and S208AGfa1p, together with the determination of possible changes in the GlcN-6-P-synthetic activity. The results of these experiments are shown in Fig. 4. It was found that incubation of GAHp-His 6 and Gfa1p with protein kinase A in the presence of ATP and cAMP, resulted in an almost two-fold increase of the amidohydrolysing activity of both proteins, closely correlated with a very similar enhancement of the synthetic activity of the whole enzyme. On the other hand, no change in both activities of S208AGfa1p, lacking the phosphorylation site, was noted. It seems therefore very likely that the enhanced GlcN-6-P-synthetic activity of Gfa1p phosphorylated at S208, is a direct consequence of the elevated amidohydrolysing activity of GAH. In order to confirm protein phosphorylation and the identity of the phosphorylation site, SDS-PAGE analysis of the reaction mixtures, followed by specific gel staining, was performed. The results of this experiment are shown in Fig. The hexosephosphate-isomerising activity of His 6 -ISOMp and the amidohydrolysing activity GAHp-His 6 were virtually unaffected by UDP-GlcNAc, known to be a feedback inhibitor of eukaryotic GlcN-6-P synthase [9 and references cited therein]. In the presence of 2 mM UDP-GlcNAc, the K m of His 6 -ISOMp for Fru-6-P was 1.5 0.1 mM and k cat was 0.53 0.04 min -1 , while the K m of GAHp-His 6 for GLUPA was 0.29 0.03 mM and k cat was 0.48 0.05 min -1 . On the other hand, UDP-GlcNAc lowered the GlcN-6-P-synthetic activity and amidohydrolysing activity of Gfa1p. For the former, the k cat value determined in the presence of 2 mM UDP-GlcNAc was 285 18 min -1 , compared to 720 24 min -1 , determined for the inhibitorfree enzyme. In the case of the amidohydrolysing activity, the effect was much stronger when Fru-6-P was also present in the incubation mixture. The k cat for GLUPA hydrolysis determined in the presence of 2 mM UDP-GlcNAc was 0.62 0.06 min -1 and in the presence of 2 mM UDP-GlcNAc + 10 mM Fru-6-P was 19.3 1.1 min - 1 , compared to 0.74 0.05 min -1 (-Fru-6-P) and 54.8 2.8 min -1 (+ Fru-6-P) determined in the absence of UDP-GlcNAc. The K m values were not affected by UDP-GlcNAc.

The gel shown in

Characterisation of 218-283Gfa1p and the UDP-GlcNAc binding site

The polypeptide chain of GlcN-6-P synthase of eukaryotic origin is generally 60 -100 aa longer than its prokaryotic counterpart. Most of the inserts responsible for this elongation are located in the GAH region, and one of them is especially long in all known sequences [START_REF] Milewski | Glucosamine-6-phosphate synthase -the multifacets enzyme[END_REF]. In the C. albicans Gfa1p, the long insert comprises the 218-283 region (see Fig. 1). In order to find any evidence for the possible role of the 218-283 part of the enzyme, we constructed its truncated version, 218-283Gfa1p. The protein was isolated and characterised. Molecular mass of the native 218-283Gfa1p was found to be 290 ± 2 kDa, while that determined under denaturing condition was roughly four times lower (74 ± 1 kDa), thus indicating that the removal of the 218-283 fragment does not affect the quaternary structure of Gfa1p.

The catalytic properties of the truncated enzyme were also characterised and compared to those of the wild-type enzyme. The measured activities are summarised in Table 3. It is evident that the truncated Gfa1p is not able to form GlcN-6-P but retains amidohydrolysing and hexosephosphate isomerising activity of the intact enzyme.

Gfa1p contains three tryptophanyl residue, namely: W97, W388 and W690. As the first one is located in the GAH domain and the remaining two in the ISOM domain, we were able to use tryptophan fluorescence to study the interaction of GAHp-His 6 , His 6 -ISOMp, 218-283Gfa1p and Gfa1p with UDP-GlcNAc, by monitoring conformational changes induced in these proteins upon ligand binding.

Emission spectra, taken at the Trp-specific excitation wavelength, were obtained and evaluated. We observed that the presence of UDP-GlcNAc strongly diminished the tryptophan-derived fluorescence of Gfa1p, thus indicating changes in the protein conformation, upon which at least one tryptophanyl residue becomes more exposed to the bulk solvent. The same effect was observed in the His 6 -ISOMp/UDP-GlcNAc spectrum, while the GAHp-His 6 fluorescence was not affected by the ligand (spectra not shown). On the other hand, changes observed in the fluorescence spectrum of 218-283Gfa1p induced by UDP-GlcNAc (not shown) were very similar to those found for the intact enzyme. The decrease in Gfa1p, 218-283Gfa1p and His 6 -ISOMp fluorescence induced by UDP-GlcNAc was ligand concentration-dependent and some subtle changes were observed even at micromolar level (data not shown), corresponding to the level of the protein concentration, thus indicating a specific ligand binding.

One may therefore conclude that in the complete Gfa1p the conformational change triggered in ISOM upon UDP-GlcNAc binding is subsequently transferred to GAH through some interdomain interactions to affect there the amidohydrolysing activity, whereas the 218-283 fragment of GAH does not seem to be involved in interactions with UDP-GlcNAc.

Interdomain communication and signalling

Structural studies on E. coli GlcN-6-P synthase revealed the presence of a 18 Å-long hydrophobic channel (see Fig. 2) between the active sites of the GAH and ISOM domains, constituted by the residues L601, A602, V605, V608 of ISOM and R26 and W74 of GAH, with the additional participation of K503 and H504 of the neighbouring protein subunit in the functional dimer. The channel allows ammonia transfer from GAH to ISOM and is gated by the indole ring of W74 [START_REF] Teplyakov | Channeling of ammonia in glucosamine-6-phosphate synthase[END_REF][START_REF] Mouilleron | Glutamine binding opens the ammonia channel and activates glucosamine-6P synthase[END_REF]. All the above mentioned residues are strongly conserved in the known GlcN-6-P synthase sequences [START_REF] Milewski | Glucosamine-6-phosphate synthase -the multifacets enzyme[END_REF] and are also present in C. albicans Gfa1p as: R32, W97, L705, A706, V709, V711, K606 and H607. In the present studies we have constructed an characterised a few recombinant versions of Gfa1p, truncated or mutated at sites supposed to be crucial for the functioning of the putative channel. Particularly, four C-terminal residues were removed to disturb the channel integrity, valine 711 was substituted by phenylalanine to block the channel and finally tryptophan 97 was exchanged for phenylalanine or glycine to reduce or remove a putative molecular gate responsible for channel opening and closure. All the constructed mutants were tested for catalytic activity.

The results are presented in Table 3. It is clear that the removal of residues 709-712 results in the complete elimination of the GlcN-6-P-synthetic activity of Gfa1p, while its hexosephosphate-isomerising activity remains unaffected. The ability to synthesis GlcN-6-P was not restored when we used ammonium chloride (10 -50 mM) and 10 mM Fru-6-P as substrates (detailed data not shown). On the other hand, substitution of W97 for phenylalanine or glycine affected the amidohydrolysing activity and GlcN-6-P formation but not the hexose phosphate isomerisation. The effect was much stronger in the case of the W97G mutant, which lost almost all synthetic activity and its ability to hydrolyse GLUPA was reduced to 10% of the original value. Interesting differences and changes in the amidohydrolysing activity, measured in the absence or presence of Fru-6-P were noted. We observed a 70 -80-fold increase of the amidohydrolysing activity of Gfa1p, V711FGfa1p, W97FGfa1p and W97GGfa1p in the presence of Fru-6-P, while that of 709-712Gfa1p and 218-283Gfa1p was only slightly enhanced. These results indicate a possibility of an interdomain signalling in C. albicans Gfa1p. It should be noted that Mouilleron et al. suggested that in the E. coli GlcN-6-P synthase, interactions between residues constituting the C-tail of ISOM, especially T606, and C1 and N98 of GAH were responsible for a substantial enhancement of the amidohydrolase activity of GAH upon Fru-6-P binding at ISOM [START_REF] Mouilleron | Glutamine binding opens the ammonia channel and activates glucosamine-6P synthase[END_REF]. Our studies thus provided evidence supporting that suggestion, since the C-tail: GAH interactions and, in consequence, the interdomain signalling, were apparently precluded upon 709-712 deletion. The negligible effect of Fru-6-P on amidohydrolysing activity of 218-283Gfa1p suggests that the interdomain signalling is also lost upon the 218-283 deletion. Rather unexpectedly, the amidohydrolysing activity of 709-712Gfa1p, measured in the absence of Fru-6-P, was nearly 25-fold higher than that of Gfa1p. This is surprising, since 709-712Gfa1p lacks a part of the C-tail, including the T710 residue, corresponding to T606 of the bacterial GlcN-6-P synthase. Possibly some new interactions between the remaining part of the C-tail and GAH, enhancing amidohydrolysing potential of GAH but practically independent of Fru-6-P binding, were created upon the removal of residues 709-712. Obviously, such an explanation is only speculative, unless the Gfa1p and 709-712Gfa1p structures are determined.

The data presented in Table 2 indicate that the K m values for the substrate in the amidohydrolysing activity of GAH-His 6 and Gfa1p were not very different. This is in contrast to the previous observation by Isupov et al., made on the bacterial GlcN-6-P synthase, where the K m for the whole enzyme was more than two orders of magnitude lower, as compared to that of the isolated domain [START_REF] Isupov | Substrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 Å structure of the glutaminase domain of glucosamine-6-phosphate synthase[END_REF]. Teplyakov et al.

suggested that the higher affinity of the E. coli whole enzyme for glutamine was due to the interdomain interactions involving the R539 residue of ISOM and the Q-loop (residues 73-80) of GAH [START_REF] Teplyakov | Channeling of ammonia in glucosamine-6-phosphate synthase[END_REF]. Our results may thus indicate that such interactions affecting the glutamine binding do not exist in C. albicans Gfa1p, in spite of the fact that the above-mentioned residues are conserved in the fungal enzyme (see Fig. 1).

Differences in the amidohydrolysing activity of the obtained muteins were of special interest. However, it must be kept in mind that the activity was measured with an artificial substrate. In order to check whether the observed phenomena take place also in the case of the physiological substrate, we measured ammonia release from L-glutamine in the absence of Fru-6-P for muteins and the wild-type enzyme. The results are shown in Fig. 6. They confirm all the previous findings concerning the amidohydrolysing activity measured with GLUPA.

The V711F mutant exhibited extremely low but measurable synthetic activity, thus indicating that the introduced phenyl ring effectively blocks the interdomain channel and thus virtually prevents ammonia transfer from GAH to ISOM. The channel blockage did not influence catalytic actions of both domains, since the measured respective activities were very much the same as those of the wild-type enzyme.

DISCUSSION

Results of our studies presented here indicate that the two-domain structure, demonstrated previously in the subunit of the bacterial GlcN-6-P synthase [START_REF] Teplyakov | Channeling of ammonia in glucosamine-6-phosphate synthase[END_REF][START_REF] Isupov | Substrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 Å structure of the glutaminase domain of glucosamine-6-phosphate synthase[END_REF][START_REF] Denisot | Glucosamine-6-phosphate synthase from Escherichia coli yields two proteins upon limited proteolysis: identification of the glutamine amidohydrolase and 2R ketose/aldose isomerasebearing domains based on their biochemical properties[END_REF][START_REF] Teplyakov | Involvement of the C-terminus in intramolecular channeling in glucosamine-6-phosphate synthase: evidence from a 1.6 Å structure of the isomerase domain[END_REF],

is also a feature of the fungal enzyme and the GAH functionality can be attributed to the 1-345 fragment of C. albicans Gfa1p, while the remaining part of the enzyme is catalytically active as ISOM. Our present results also show that formation of the previously suggested homotetrameric structure of C. albicans GlcN-6-P synthase [START_REF] Milewski | Oligomeric structure and regulation of Candida albicans glucosamine-6-phosphate synthase[END_REF][START_REF] Olchowy | Crystallization and preliminary X-ray analysis of the isomerase domain of glucosamine-6-phosphate synthase from Candida albicans[END_REF] is mediated by interactions between the ISOM domains, while the GAH domains do not seem to be involved in the oligomerisation. Thus the quaternary structure of this enzyme is clearly different from that of its bacterial, homodimeric counterpart, as well as from that of the "sister" class II amidotransferase, namely L- glutamine: phosphoribosylpyrophosphate amidotransferase, for which it was previously found that the formation of the tetrameric structure was mediated by both domains [START_REF] Muchmore | Crystal structure of glutamine phosphoribisylpyrophosphate amidotransferase from Escherichia coli[END_REF].

Previous studies on fungal GlcN-6-P synthase provided kinetic evidence that the UDP-GlcNAc binding site is different from the L-Gln and D-Fru-6-P binding sites [START_REF] Milewski | Oligomeric structure and regulation of Candida albicans glucosamine-6-phosphate synthase[END_REF][START_REF] Endo | Feedback inhibition of L-glutamine Dfructose 6-phosphate amidotransferase by uridine diphosphate Nacetylglucosamine in Neurospora crassa[END_REF]. Our present results demonstrate that the inhibitor binding site is located at ISOM and the binding of UDP-GlcNAc induces conformational changes in this domain. Such location of the UDP-GlcNAc binding site(s) confirms suggestions made by Chou for the mammalian GlcN-6-P synthase, based on in silico homology modelling [START_REF] Chou | Molecular therapeutic target for type-2 diabetes[END_REF]. Our current results also show that despite the detected conformational changes, the UDP-GlcNAc binding to Gfa1p does not affect the hexosephosphate-isomerising activity of ISOM but lowers the amidohydrolysing activity of GAH and, in consequence, the GlcN-6-P-synthetic activity of the entire enzyme. A structural basis of this effect remains unclear until the exact location of the UDP-GlcNAc binding site in ISOM is known.

Regulation of the GlcN-6-P synthase activity by enzymatic phosphorylation and dephosphorylation was previously demonstrated for the mammalian and fungal enzymes [14,25 -27]. Our present results prove that an increase of GlcN-6-P synthetic activity of the C. albicans Gfa1p, observed upon phosphorylation of the S208 residue of this enzyme, is most likely due to the enhancement of the GAH amidohydrolysing activity. The molecular mechanism of this effect remains unknown, until a structure of the fungal enzyme is solved. However, it seems worth mentioning that in the 3D structure of the bacterial enzyme, the region corresponding to the kinase A recognition site in the eukaryotic GlcN-6-P synthase, is located close to the catalytic C1 residue. Particularly, the hydroxyl group of S176 (homologous to S208 of the C. albicans enzyme), is only 5.4 Å from the protonated -amino group of C1 [START_REF] Teplyakov | Channeling of ammonia in glucosamine-6-phosphate synthase[END_REF][START_REF] Mouilleron | Glutamine binding opens the ammonia channel and activates glucosamine-6P synthase[END_REF][START_REF] Isupov | Substrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 Å structure of the glutaminase domain of glucosamine-6-phosphate synthase[END_REF]. If the same is true for the fungal enzyme, one may assume that the phosphorylated S208 may help keep C1 in the active conformation. However, the fungal GAH contains several inserts in the 1-208 region, absent from the bacterial enzyme, which may also affect the relative positioning of C1 and S208.

A possible role of the 218-283 fragment in C. albicans Gfa1p remains one of the particularly intriguing issues. Similar inserts, smaller or slightly larger, are present in all known eukaryotic sequences, although they demonstrate very little, if any, sequence homology [START_REF] Milewski | Glucosamine-6-phosphate synthase -the multifacets enzyme[END_REF], that could suggest that the respective regions may not have any particular function. Our present results indicate that the 218-283 fragment is neither involved in UDP-GlcNAc binding nor in the formation of the quaternary structure. The fact that the 218-283Gfa1p lacks the GlcN-6-P-synthetic activity can be, in our opinion, interpreted as a consequence of a possible improper relative orientation of GAH and ISOM that prevents the assembly of the interdomain channel and thus precludes the ammonia transfer from GAH to ISOM. This suggestion seems to be additionally supported by the observed disturbance of the interdomain signalling.

The loss of the GlcN-6-P-synthetic activity of Gfa1p upon removal of the four C-terminal residues, indicates that the integrity of the interdomain channel is crucial for the ammonia transfer from GAH to ISOM. Therefore, it is very likely that the channel in 709-712Gfa1p is leaky and ammonia molecules, generated from Lglutamine at the GAH active site, do not reach the ISOM active site but are released to the bulk solvent. On the other hand, the escape route created by the 709-712 deletion seems to be unidirectional, since the truncated enzyme was still unable to use exogenous ammonia as the substrate for GlcN-6-P formation. However, one must keep in mind that GlcN-6-P synthase, unlike some other amidotransferases [START_REF] Massière | The mechanism of glutaminedependent amidotransferases[END_REF],

does not possess any separate binding site for ammonia. Therefore, the NH 3 molecules from the bulk solvent, randomly interacting with enzyme molecules, may have little chance to enter the ISOM active site from a proper direction.

Finally our present results provide evidence for the crucial role of the W97 residue which is a counterpart of W74 of the E. coli enzyme. In our W97F and W97G mutants, this putative molecular gate was modified: reduced in the former or virtually eliminated in the latter. These modifications have been shown to substantially decrease the GlcN-6-P-synthetic activity of the enzyme, by an order and more than two orders of magnitude, respectively. In our opinion this phenomenon may be well explained in light of the previous observations of Mouilleron et al., who found that the indole ring of W74 in E. coli enzyme, after rotation induced upon glutamine binding, opened the channel and at same time sealed its side wall [START_REF] Mouilleron | Glutamine binding opens the ammonia channel and activates glucosamine-6P synthase[END_REF]. We assume therefore, that the channel in the W97F W97G muteins of Gfa1p is leaky to some extent, resulting in lower efficiency of ammonia transfer from GAH to ISOM. Interestingly enough, both substitutions resulted also in a slight decrease of the amidohydrolysing activity. This effect might be in turn caused by the fact that W74 in E. coli GlcN-6-P synthase, and possibly its counterparts in other enzyme variants, are parts of the Qloop (residues 73-80 in the E. coli enzyme; 98-103 in the C. albicans homologue), functioning as a lid that closes the entrance to the GAH active site when L-glutamine is bound [START_REF] Teplyakov | Channeling of ammonia in glucosamine-6-phosphate synthase[END_REF][START_REF] Mouilleron | Glutamine binding opens the ammonia channel and activates glucosamine-6P synthase[END_REF]. The W97F and W97G substitutions may distort the lid structure, thus making it less efficient as a "substrate keeper". The V711F substitution appeared to create an additional molecular gate in the interdomain channel. This effect was in fact expected, as comparison of the structures of channel segments in V711F and the wild-type enzyme, made by molecular homology modelling (Fig. 7), revealed that the phenyl ring should provide a steric hindrance to the channel. A similar phenomenon had been observed in the G359Y and G359F mutants of E. coli carbamoyl phosphate synthetase (CPS) [START_REF] Thoden | Carbamoylphosphate synthetase. Creation of an escape route for ammonia[END_REF][START_REF] Huang | An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthase prevents the use of glutamine as a substrate but not ammonia[END_REF], in the L109A mutant of E. coli CTP synthase [START_REF] Lunn | Alternative substrates for wild-type and L109A E. coli CTP synthases. Kinetic evidence for a constricted ammonia tunnel[END_REF] and in the L415A mutant of glutamine phosphoribosylpyrophosphate amidotransferase [START_REF] Bera | Dual role for the glutamine phosphoribosylpyrophosphate amidotransferase ammonia channel. Interdomain signaling and intermediate channeling[END_REF]. In all those cases the partial reactions of glutamine hydrolysis and the final product formation became uncoupled, but both enzymes remained able to use exogenous ammonia as the substrate. It was postulated that the molecular channel in bacterial CPS becomes leaky upon Gly 359 substitution with Tyr or Phe [START_REF] Huang | An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthase prevents the use of glutamine as a substrate but not ammonia[END_REF], while the channel in the CTP synthase was constricted upon the L109A exchange [START_REF] Lunn | Alternative substrates for wild-type and L109A E. coli CTP synthases. Kinetic evidence for a constricted ammonia tunnel[END_REF]. Our V711F mutant did not gain any ability to use ammonia for GlcN-6-P formation, but the partial activities were not affected. It seems therefore that in the V711F mutant, L-glutamine or L-glutamyl-p-nitroanilide is hydrolysed at GAH and the respective product, namely ammonia or p-nitroanilide, is released to the bulk solvent.

In conclusion, we were able to identify the separately expressed 1-345 and 346-712 fragments of C. albicans Gfa1p as the glutamine amide-hydrolysing domain (GAH) and the hexose phosphate-isomerising domain (ISOM), respectively. The interactions between the ISOM domains are responsible for the formation of a homotetrameric structure of the native Gfa1p, while GAH is not involved in protein oligomerisation. The binding site for the feedback inhibitor, UDP-GlcNAc, is located in the ISOM domain. Binding of UDP-GlcNAc at ISOM or phosphorylation of the S208 residue affects the amidohydrolysing activity of GAH and, in consequence, the GlcN-6-P synthetic activity of the whole enzyme. No specific function can be attributed to the 218-283 fragment of GAH, however its deletion strongly disrupts the interdomain communication and signalling. A similar effect is observed upon the removal of four C-terminal residues. The intramolecular channel connecting GAH and ISOM is constituted by several hydrophobic residues and one of them, namely W97, functions as a molecular gate. The channel becomes leaky upon the deletion of amino acids 709-712, as well as upon the reduction of the molecular gate size. On the other hand, the channel can be blocked upon the V711F substitution. Functional domains, some relevant fragments and the intramolecular channel (small spheres) are indicated. The image has been generated from 1JXA [START_REF] Teplyakov | Channeling of ammonia in glucosamine-6-phosphate synthase[END_REF]. Figure 7 The blockage of the interdomain channel in Gfa1p upon V711F substitution.

(A) Computed structure created by homology modelling using E. coli GlcN-6-P synthase (1JXA) [START_REF] Teplyakov | Channeling of ammonia in glucosamine-6-phosphate synthase[END_REF] as a template. (B) Putative result of V711F substitution.

Residues marked with an asterisk are from the neighbouring subunit. The channel is indicated by black spheres.

  ttc att caa ttc cat ttc taa agt ttg-3'. The artificial cloning sites: KpnI, NdeI (GLN1-KN) and XhoI, HindIII (GLN2-HX), at the 5' ends of primers (underlined) were introduced to facilitate the cloning procedure. The PCR product (1071 bp) purified from an agarose gel band was cloned directionally into KpnI and HindIII sites of the pUC19 vector and then re-cloned into the pET23b plasmid, giving a recombinant expression plasmid pET23b-GLN /4628 bp/. The identity of the plasmids was confirmed by restriction analysis and DNA sequencing. The obtained construct encoded the putative GAH domain of C. albicans GlcN-6-P synthase, containing an additional octapeptide (LEHHHHHH) at the C-terminus (GAHp-His 6 ).Analogously, the respective GFA1 gene fragment encoding residues of Gfa1p 356-712 was amplified from the pUC19-CaGFA1 plasmid by PCR using the following primers: Fru1-BNK, 5'-cct ggc gga tcc cat atg cac cac cac cac cac cac ggt acc atg aaa ggc ccc tat aaa cat ttt atg -3' and Fru2-Eco, 5'-cct gcc gaa ttc tta ctc aac agt aac tga ttt agc ca -3'. The artificial cloning sites: BamHI and NdeI (Fru1-BNK) and EcoRI (Fru2-Eco), at the 5' ends of primers (underlined) were introduced to facilitate the cloning procedure. After cloning into pUC19 and subsequent re-cloning into pET23b, a recombinant expression plasmid pET23b-FRU /4755 bp/ was obtained. The construct encoded the putative ISOM domain of C. albicans GlcN-6-P synthase containing an additional nonapeptide (MHHHHHHGT) at the N-terminus (His 6 -ISOMp).

9 Biochemical

 9 -6-P synthase preparation in 50 mM Tris-HCl buffer, pH = 7.5, were incubated at 25 C . The reduction of NADP + to NADPH was monitored spectrophotometrically at 340 nm, assuming Journal Immediate Publication. Published on 19 Feb 2007 as manuscript BJ20061502 © 2007 Biochemical Society 340 (NADPH) = 6 220 M -1 cm -1

  Amino acid sequences of E. coli and C. albicans GlcN-6-P synthases were aligned and the result is shown in Fig. 1. The sequences demonstrate 36% identity and 52% similarity, however several gaps, indicating regions which are absent from the shorter, bacterial variant of the enzyme, are clearly shown. Most of them are present in the N-terminal 1-239 part of the bacterial protein, constituting the GAH domain, with the largest one corresponding to the 218-283 region of the C. albicans enzyme.

  Fig 5A was stained with the GelCode kit, allowing a specific detection of the O-phosphorylated serine residues. The stained bands can be clearly seen in lanes 1, 4 and 5, thus confirming the presence of an O-phosphorylated serine in the kinase A-treated GAHp-His 6 and Gfa1p. Presence of Fru-6-P in the incubation mixture did not affect Gfa1p phosphorylation. A specific kinase A inhibitor, H-89, almost stopped phosphorylation of GAHp-His 6 (very faint band in lane 3). No band can be seen in lane 6, thus confirming that S208 is indeed the residue phosphorylated by kinase A in Gfa1p.

Table 1 .Table 2 .

 12 PCR primers used for the construction of site-directed mutants of GFA1. Restriction recognition sites are underlined. Capital letters indicate mismatches with the original GFA1 sequence (bold -introducing mutation; normal -introducing restriction recognition site).N-and C-terminal flanking oligonucleotidesGln1-KN: 5'-cctggcggtacccatatgtgtggtatttttggttacg-3' cgfa-bam: 5'-cacgggatccttactcaacagtaactgatttagcca-3Kinetic parameters of reactions catalysed by Gfa1p, its oligoHis-tagged variants and oligoHis-tagged domains.Data are the means of three independent determinations S.D. ND -

Figure 1

 1 Figure 1 The alignment of amino acid sequences of C. albicans and E. coli GlcN-6-P synthases. The linker between the GAH and ISOM domains of E. coli GlcN-6-P synthase and residues substituted in the recombinant versions of the C. albicans enzyme, constructed for the purpose of this work, are indicated. C. albicans numbering is shown.

Figure 2

 2 Figure 2 Stereo view of the 3D structure of E. coli GlcN-6-P synthase.

Figure 3 Figure 4

 34 Figure 3 SDS-PAGE analysis of the purification of N-oligoHis-tagged 346-712 fragment of Gfa1p overproduced in E. coli. Lane M, molecular mass markers; lane 1, total lysate of E. coli BL21(DE3)pLysS pET23b-FRU cells; lane 2, total lysate of control E. coli BL21(DE3)pLysS pET23b cells; lanes 3-4, wash fractions with buffer W; lane 5, fraction eluted with 0.5 M imidazole

Figure 5

 5 Figure 5 SDS-PAGE analysis of GAHp-His 6 ,Gfa1p and S208AGfa1p phosphorylation by kinase A. Mixtures obtained after 30 min incubation of a given protein with indicated components were separated by SDS-PAGE. (A) Gel stained with the GelCode kit; (B) The same gel stained with Coomassie Brilliant Blue. Lane 1, GAHp-His 6 + kinase A; lane 2, GAHp-His 6 ; lane 3, GAHp-His 6 + kinase A + H-89; lane 4, Gfa1p + kinase A; lane 5, Gfa1p + kinase A + 10 mM Fru-6-P; lane 6,

Figure 6

 6 Figure 6 The time course of ammonia release from L-glutamine, catalysed by Gfa1p and its mutated versions. Enzyme samples were incubated with 10 mM L-Gln and the released ammonia was quantified with the Nessler's reagent. Experiments were done in triplicate. Bars represent S.D.

13 Biochemical Journal Immediate Publication. Published on 19 Feb 2007 as manuscript BJ20061502 © 2007 Biochemical Society

Table 3 .

 3 Enzymatic activities of Gfa1p, its truncated and mutated versions.Data are the means of three independent determinations S.D.

		Amidohydrolysing	Hexosephosphate	GlcN-6-P-
	Enzyme version	activity	isomerising	synthetic activity
		[mU/mg] -Fru-6-P + Fru-6-P	[U/mg] activity	[U/mg]
	Gfa1p (wild type)	12 0.6	890 24	63.1 1.9	12 0.5
	218-283Gfa1p	11.6 0.2	12.5 0.7	61 2.2	<0.01
	V711F	13 0.5	940 18	62.6 1.3	0.021 0.003
	709-712	290 6	357 11	60.7 1.4	<0.01
	W97F	4.3 0.3	256 8	62.1 1.2	1.20 0.06
	W97G	1.3 0.1	95 2	63.2 1.4	0.05 0.01

Biochemical Journal Immediate Publication. Published on 19 Feb 2007 as manuscript BJ20061502 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 19 Feb 2007 as manuscript BJ20061502 © 2007 Biochemical Society

Acknowledgement

The authors acknowledge the financial support of these studies from the Polish Ministry of Science and Higher Education (grant No P06A 036 25) and in part from the Faculty of Chemistry, Gda sk University of Technology. Thanks are given to Dr.

Marek Wojciechowski for his help in molecular modelling.