Structure of Escherichia coli 5'-Methylthioadenosine/ S-Adenosylhomocysteine Nucleosidase Inhibitor Complexes Provide Insight into the Conformational Changes Required for Substrate Binding and Catalysis*

Jeffrey E. LeeDagger §, Kenneth A. Cornell||**, Michael K. Riscoe||**Dagger Dagger , and P. Lynne HowellDagger §§§

From Dagger  Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, the § Department of Biochemistry, Faculty of Medicine, University of Toronto, Medical Sciences Building, Toronto, Ontario M5S 1A8, Canada, the  Department of Chemistry, Portland State University, Portland, Oregon 97207, the || Medical Research Service, 151-0, Veterans Affairs Medical Center, Portland, Oregon 97021, and the Dagger Dagger  Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201

Received for publication, October 22, 2002, and in revised form, December 16, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is a key enzyme in a number of critical biological processes in many microbes. This nucleosidase catalyzes the irreversible hydrolysis of the N9-C1' bond of MTA or AdoHcy to form adenine and the corresponding thioribose. The key role of the MTA/AdoHcy nucleosidase in biological methylation, polyamine biosynthesis, methionine recycling, and bacterial quorum sensing has made it an important antimicrobial drug target. The crystal structures of Escherichia coli MTA/AdoHcy nucleosidase complexed with the transition state analog, formycin A (FMA), and the nonhydrolyzable substrate analog, 5'-methylthiotubercidin (MTT) have been solved to 2.2- and 2.0-Å resolution, respectively. These are the first MTA/AdoHcy nucleosidase structures to be solved in the presence of inhibitors. These structures clearly identify the residues involved in substrate binding and catalysis in the active site. Comparisons of the inhibitor complexes to the adenine-bound MTA/AdoHcy nucleosidase (Lee, J. E., Cornell, K. A., Riscoe, M. K., and Howell, P. L. (2001) Structure (Camb.) 9, 941-953) structure provide evidence for a ligand-induced conformational change in the active site and the substrate preference of the enzyme. The enzymatic mechanism has been re-examined.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The enzyme 5'-methylthioadenosine (MTA)1/S-adenosylhomocysteine (AdoHcy) nucleosidase (EC 3.2.2.9) is a dual substrate specific enzyme that irreversibly cleaves the N9-C1' bond of 5'-methylthioadenosine and S-adenosylhomocysteine to form adenine, and 5'-methylthioribose and S-ribosylhomocysteine, respectively (1).

MTA/AdoHcy nucleosidase has been described as an excellent target for broad-spectrum antimicrobial drug design (2, 3). The enzyme is not found in mammalian cells but is found in many microbial pathogens, such as Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis, Haemophilus influenza, Vibrio cholerae, and Bacillus anthracis. Inhibition of MTA/AdoHcy nucleosidase should selectively target the pathogenic microbes while leaving the human host unharmed by increasing cellular levels of MTA and AdoHcy. The buildup of these molecules will affect four major cellular functions in microbes. First, the enzyme is important in the recycling of methionine, an essential amino acid that is energetically expensive to synthesize (2, 3). Second, the nucleosidase is involved in the regulation of biological methylation, as AdoHcy is a potent negative feedback inhibitor of methyltransferases (4). Biological methylation is critical in all organisms and is responsible for regulating a number of biological processes including DNA and protein metabolism (5). Third, MTA/AdoHcy nucleosidase participates in the regulation of polyamine biosynthesis (6, 7). The role of polyamines is not well understood but they are thought to be important in growth processes. MTA acts as a potent negative feedback inhibitor of spermidine synthase. Finally, the nucleosidase has recently been implicated in the quorum sensing pathway in bacteria (8). Quorum sensing is the phenomenon whereby the accumulation of small exported organic molecules called autoinducers enables a bacterial cell to sense the population of bacteria. This phenomenon was first described in the bioluminescent marine bacterium Vibrio fischeri (9-11) and has been implicated in the regulation of virulence factors (12-14) and biofilm formation in many bacteria (15). When a threshold level of autoinducers is reached, bacteria trigger a signal transduction cascade that can alter gene expression (16). MTA/AdoHcy nucleosidase cleaves AdoHcy to generate S-ribosylhomocysteine and adenine. The enzyme LuxS acts on S-ribosylhomocysteine to form the autoinducer-2 furanone and homocysteine (8, 17). Autoinducer-2 has been implicated in intra- and interspecies communication. Inhibition of the nucleosidase is thought to prevent the formation of the product (S-ribosylhomocysteine) needed for LuxS to catalyze the production of autoinducer-2.

Previously we determined the 1.9-Å resolution crystal structure of MTA/AdoHcy nucleosidase complexed with adenine bound in the active site (18). The enzyme has a mixed alpha /beta structure with a central 9-stranded beta -sheet surrounded by six alpha  helices and a 310 helix. Although the primary sequence is not homologous to any known proteins, its tertiary structure is similar to enzymes in the purine nucleoside phosphorylase family.

This paper reports the crystal structures of MTA/AdoHcy nucleosidase complexed with the inhibitors, formycin A (FMA) and 5'-methylthiotubercidin (MTT) (Fig. 1). These inhibitors have a Ki of 10 and 0.75 µM, respectively (Fig. 1) (19). These are the first nucleosidase structures complexed with a transition state and nonhydrolyzable substrate analogs. The structures presented here reveal the residues involved in nucleoside binding and new insight into the identity of the catalytic base. Comparisons of the adenine-bound and the inhibitor-bound MTA/AdoHcy nucleosidase structures provide evidence of inter- and intrasubunit conformational changes in the active site. These conformational changes are required for substrate binding and catalysis. Furthermore, these comparisons have allowed a structural explanation of the substrate preference of the enzyme toward 6-aminopurine nucleosides, an intact ribose, and a 5'-neutral sulfur atom (19). Taken together, these structures have enabled us to re-examine the catalytic mechanism involved in the cleavage of the N9-C1' bond.


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Fig. 1.   Structures of MTA, FMA, and MTT. Please note that for ease of comparison, the numbering of atoms in the purine base of the inhibitors (MTT and FMA) is based on the numbering convention of MTA and not the IUPAC standard.


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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Protein Preparation and Crystallization-- The expression and purification of Escherichia coli MTA/AdoHcy nucleosidase was reported earlier (20). Commercial sparse matrix screens from Hampton Research and Emerald Biostructures were set up using purified protein (15 mg/ml) incubated with 1 mM FMA or 1 mM MTT on ice for 2 h. Rod-shaped crystals (0.15 × 0.15 × 1.0 mm) were grown at room temperature by the hanging drop vapor diffusion technique over a 3-week period using 37% (w/v) PEG 200, 100 mM sodium acetate, pH 4.7, 100 mM NaCl, and 8.0 mM cobalt chloride.

Data Collection and Processing-- The FMA and MTT nucleosidase co-crystals were frozen without use of further cryoprotectant in a stream of nitrogen gas (100 K). Data for the nucleosidase-FMA complex were measured on a R-AXIS-IV++ image plate and RUH3R rotating anode x-ray generator with Osmic optics. Data for the MTT containing crystal were measured at Station X8C at the National Synchrotron Light Source (Brookhaven National Laboratories). All data were processed using the program d*TREK (21). The data collection statistics are presented in Table I.

Structural Determination and Refinement-- The program CNS (22) was used for all structure determination and refinement steps. The FMA-bound nucleosidase structure was determined by molecular replacement using the adenine-bound E. coli MTA/AdoHcy nucleosidase (Protein Data Bank code 1JYS) dimer as the search model. Data between 12- and 5-Å resolution were included in the cross-rotation and translation functions. The resulting model was then refined using torsion angle simulated annealing. The interactive computer graphics program Xfit in the XtalView crystallographic suite (23) was used to rebuild the initial FMA-containing model. Several rounds of torsion angle simulated annealing (24) starting at 5000 K using all data with no sigma  cutoff and a bulk solvent correction were alternated with manual rebuilding in Xfit. The progress of the refinement was monitored by reductions in Rcryst and Rfree factors. Difference Fourier maps calculated after the initial round of simulated annealing revealed strong positive electron density corresponding to FMA in the two active sites. The FMA coordinates were generated by modifying the Protein Data Bank file of adenosine downloaded from the HIC-UP server (25). The topology and parameter files for FMA were generated using the XPLO2D server (26). Water molecules were included into the model during the later rounds of the refinement based on the presence of a 3sigma peak in the sigma A-weighted Fo - Fc difference electron density maps and at least one hydrogen bond to a protein, inhibitor, or solvent atom. After addition of the inhibitor and water molecules, alternating rounds of crystallographic conjugate gradient minimization refinement and model rebuilding in Xfit were performed.

Given that the space group and cell constants of the MTT and FMA-bound nucleosidase were isomorphous, the MTT-bound nucleosidase structure could likely have been solved by difference Fourier methods. However, as an additional verification of our previous result, molecular replacement was employed with the FMA-complexed dimer as a search model. Data between 12- and 4-Å resolution were included in the cross-rotation and translation functions. The resulting model was subjected to a round of rigid-body refinement using all data between 35 and 2.0 Å in CNS. sigma A-Weighted difference and 2Fo - Fc Fourier maps were calculated and the model was rebuilt using Xfit. The model was subsequently refined using torsion angle-simulated annealing and conjugate gradient minimization refinement, as described above. The MTT inhibitor was visible as the largest peak in the difference Fourier maps. The MTT molecule was generated by modifying the coordinates of the MTA ligand in the MTA phosphorylase structure (Protein Data Bank code 1CG6) (27). The topology and parameter files were generated using the XPLO2D server (26). Analysis of both structures using PROCHECK (28) reveal that none of the nonglycine residues fall into the disallowed region of the Ramachandran plot. The refinement statistics for the structures are reported in Table I.

Superimposition of Structures-- The MTA/AdoHcy nucleosidase structures were aligned by nonlinear least-squares fit of selected main chain (N-Calpha -C) atoms in the central beta -sheet using the program PROFIT (version 6.0), written by G. David Smith. The residue ranges used in the refinement of the superposition were Lys2-Ile6, Glu42-Leu46, Leu62-Ala77, Val90-Arg96, Pro116-Ala121, and Val187-Val191.

Protein Data Bank Accession Codes-- The coordinates of the formycin A and MTT complexes have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data bank (29), Protein Data Bank codes 1NC3 and 1NC1, respectively.

    RESULTS AND DISCUSSION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Structural Determination

The nucleosidase used for crystallization consists of 232 amino acids and a 10-residue N-terminal extension. The 10-residue N-terminal extension is part of a larger 31-amino acid 6-histidine fusion tag used for purification. Chymotrypsin digestion of the enzyme results in the cleavage of the first 21 residues of the 31-residue tag. In the FMA and MTT nucleosidase structures, a dimer is present in the asymmetric unit. In both structures all 232 residues of the protein were clearly visible in the electron density map and have been modeled. The FMA and MTT complexed structures were refined to a Rcryst = 19.8% and Rfree = 24.7%, and Rcryst = 19.0% and Rfree = 22.3%, respectively (Table I). Analysis of the structures in PROCHECK (28) reveal that none of the nonglycine residues fall into the disallowed region of the Ramachandran plot.

                              
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Table I
Data collection and refinement statistics

Overall Structure

In the FMA and MTT complexed MTA/AdoHcy nucleosidase structures, two monomers related by 2-fold noncrystallographic symmetry are present in the asymmetric unit (Fig. 2a). This is consistent with our observation that the nucleosidase elutes from an analytical fast protein liquid chromatography size exclusion column (Superdex-75 HR 10/30) with a retention time equivalent to a 51-kDa protein (18). The overall topology of the FMA and MTT nucleosidase complexes is similar to that previously published for the enzyme complexed with adenine (18). Each subunit has a central 9-stranded mixed beta -sheet and a smaller 5 beta -stranded sheet surrounded by six alpha -helices and a small 310 helix (Fig. 2b). The root mean square deviation (r.m.s.d.) between monomers A and B in the FMA- and MTT-bound structures are 0.20 and 0.22 Å, respectively. Strong electron density was seen in both active sites of the MTA/AdoHcy nucleosidase structures throughout refinement indicating that each active site is fully occupied with one bound inhibitor (Fig. 2, c and d).


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Fig. 2.   E. coli MTA/AdoHcy nucleosidase inhibitor complexes. Ribbon diagram of the overall structure of the MTT bound dimer viewed down the noncrystallographic 2-fold axis (a) and the MTT complexed monomer (b). The ribbon diagrams were generated using Swiss-PDB Viewer (35) and POV-RAY. Stereodiagram of the initial E. coli MTA/AdoHcy nucleosidase sigma A-weighted  Fo  -  Fc  electron density map superimposed with the refined FMA-bound (c) and MTT-bound (d) nucleosidase models. The 3sigma contoured electron density maps were created using Xfit (23).

Active Site

The active site of MTA/AdoHcy nucleosidase was previously identified from the adenine-bound nucleosidase complex and was suggested to comprise two parts, an adenine- and a ribose-binding site (18). Given that only a weakly bound adenine molecule was present in the active site of the previous structure, we were only able to define the adenine-binding site with any certainty. Modeling MTA into the active site putatively identified the residues involved in the ribose-binding site. Examination of the FMA and MTT complexes now suggest that the active site can be divided into three regions: 1) the adenine, 2) the ribose, and 3) the 5'-alkylthio-binding sites. Please note that for ease of comparison, the numbering of atoms in the purine base of the inhibitors FMA and MTT will be based on the numbering convention of MTA and not the IUPAC standard (Fig. 1).

Adenine-binding Site-- The adenine-binding site is a deep pocket formed by beta 10 and a loop between beta 8 and alpha 4. Based on the FMA and MTT complexed structures (Figs. 3 and 4), this site consists of residues Phe151, Ile152, Ser196, Asp197, and Ala199. Phe151 makes a base-stacking interaction with the adenine ring. An aromatic residue (Phe or Tyr) is structurally conserved between all known MTA/AdoHcy nucleosidases and the purine nucleoside phosphorylase family of enzymes. The main chain carbonyl oxygen and amide nitrogen of Ile152 are in excellent hydrogen bonding distance to the amino group (N6) and N1 of the adenine base, respectively. Additional interactions exist between Odelta 2 and Odelta 1 of Asp197 and the N7 and N6 amino group of the adenine base, respectively, in the FMA-bound nucleosidase structure. In the MTT-complexed model only a hydrogen bond interaction between Odelta 1 of Asp197 and the N6 amino group exists, the N7 purine base position is replaced by a carbon in MTT. Ser196 and Ala199 do not interact with the inhibitor directly but are involved in orienting the side chain of the putative catalytic acid, Asp197. The Ser196 Ogamma and amide nitrogen of Ala199 help anchor the Asp197 side chain by making hydrogen bonds to the Odelta 2 and Odelta 1 of Asp197, respectively. In the FMA-bound structure, N8 makes an additional hydrogen bond to the Ogamma hydrogen of Ser76.


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Fig. 3.   Active site of MTA/AdoHcy nucleosidase. Adenine-binding site with FMA (a) and MTT (b) bound. Ribose and 5'-alkylthio-binding site with complexed FMA (c) and MTT (d) molecules. Residues colored in orange are donated from the neighboring subunit. e, Glu12 hydrogen bonding patterns in monomers A and B of the FMA-bound nucleosidase active site. These ball and stick representations were generated using VMD (36).


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Fig. 4.   Active site schematic of MTA/AdoHcy nucleosidase. A schematic of the interactions made between the nucleosidase and FMA (a) in monomers A and B, and MTT (b). Dotted lines represent protein-protein or protein-ligand hydrogen bonds with distances in angstroms (Å). Residues donated from a neighboring subunit are shown in shaded boxes. In panel b, the numbers in and outside the brackets refer to hydrogen bonding distances in monomers A and B, respectively.

Ribose-binding Site-- The ribose-binding site is primarily made up of six residues, Met9, Ser76, Met173, Glu174, Arg193, and Phe207 (Figs. 3 and 4). Previously, only residues Met173, Glu174, and Arg193 were proposed to be involved in saccharide binding. The ribose in both the FMA and MTT complexed structures are anchored by both hydrogen bonds and van der Waals contacts. The hydrogen-bonding network consists of four direct and one water-mediated hydrogen bond to the enzyme. Two hydrogen bonds are made by the Oepsilon 1 and Oepsilon 2 of Glu174 to the O3' and O2' hydroxyls of the ribose, respectively. A third hydrogen bond is made from the backbone amide hydrogen of Met173 to the O2' hydroxyl of the ribose, whereas the hydrophobic side chain of Met173 packs against the hydrophobic face of the ribose moiety. This interaction is thought to help stabilize and properly orient the sugar. The fourth direct hydrogen bond is made between the Ogamma hydrogen of Ser76 and the O4' oxygen of the ribose. The water-mediated hydrogen bond interactions are between WAT3 and the O2' and O3' ribosyl hydroxyls. van der Waals contacts are made between Met9 Sdelta and Cepsilon and the O4' and C4' positions of the ribose, respectively. In addition, Phe207 Czeta makes a van der Waals contact to the C4' position.

5'-Alkylthio-binding Site-- The 5' substituents of the inhibitors are primarily coordinated by hydrophobic interactions. This hydrophobic pocket is made up of residues Met9, Ile50, Val102, Phe105, Pro113, Phe151, Met173, and Phe207 (Figs. 3 and 4). Previously, we had suggested that residues Ile50, Val102, Phe105, Tyr107, Pro113, Phe151, and Phe207 would make van der Waals-type interactions with the 5'-alkylthio group, with residues Val102, Phe105, Tyr107, and Pro113 being donated from a neighboring subunit (Fig. 3, c and d, in orange). The FMA- and MTT-bound structures confirm the participation of the previously proposed residues in the binding of the 5' moiety with three exceptions. The 5'-alkylthio binding pocket also includes residues Met9 and Met173, as these residues form van der Waals type interactions to the 5'-methylthio group. The hydrophobic binding pocket does not include Tyr107 in the presence of MTT, FMA, or MTA. In the FMA- and MTT-complexed structures, Tyr107 is ~6 Å away from the 5'-methyl moiety. Although Tyr107 is too far to play a major role in the binding of the methylthio group, the Oeta of Tyr107 may be engaged in a hydrogen bond with the carboxylic acid or amino group of the homocysteinyl moiety of AdoHcy.

Conformation of the Nucleosides-- In solution, MTA and AdoHcy are found predominantly in the anti-conformation and exhibit a glycosidic torsion angle of 45° (C8-N9-C1'-O4') (30). The FMA and MTT nucleosides bound to MTA/AdoHcy nucleosidase are found in a high syn-conformation relative to the glycosidic bond. This energetically unfavorable orientation of the substrate puts strain on the substrate and presumably favors the cleavage of the N9-C1' bond. In the human MTAP structure, the bound MTA also exhibited a high syn-conformation with a glycosidic torsion angle of -54° (27). In the FMA- and MTT-bound structures, FMA and MTT have glycosidic torsion angles of -68° and -64°, respectively. The superimposition of the enzyme-bound MTA, FMA, and MTT structures confirm the similarity in the glycosidic torsion angle (Fig. 5). However, the superimposition also reveals differences in the torsion angles of the 5'-alkylthio tail. MTA bound in the human MTAP structure has a 5'-alkylthio torsion angle (C4'-C5'-S5-CS) of 39°, whereas MTT in the nucleosidase has a torsion angle of -83° (Fig. 5).


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Fig. 5.   Conformations of the MTA, MTT, and FMA nucleosides in the active site. The MTT (red) and FMA (green) in the inhibitor-bound structures were superimposed with MTA (blue) from human MTA phosphorylase (Protein Data Bank code 1CG6) (27). The figure was generated using VMD (36).

Conformational Changes

Three structures of E. coli MTA/AdoHcy nucleosidase have now been determined, and the protein complexed with adenine, FMA, and MTT. A detailed comparison of these structures has been carried out to determine whether any conformational changes occur on substrate binding. The structures were superimposed using residues in the central beta -sheet as described under "Experimental Procedures." A superimposition of the dimeric FMA and MTT inhibitor complexes based on the beta -sheets from both subunits revealed an overall r.m.s.d. of 0.3 Å for the main chain atoms (N-Calpha -C). The two structures are virtually identical and for the purpose of the following discussion, we have used the MTT complex as the representative model to describe the structural changes observed. A r.m.s.d. plot of all Calpha atoms in the monomer of the adenine- and MTT-complexed structures highlights six major regions of the structure that deviate by more than 2.0 Å (labeled 1-6 in Fig. 6a). However, these six regions can be simplified into three areas of conformational change, as changes in region 1 are propagated to regions 4 and 6 and region 2 affects region 5. As an additional check on the validity of our superimposition, the adenine-bound structure was superimposed onto the MTT-complexed structure based on the adenine moiety. The r.m.s.d. plot of the Calpha positions shows similar regions of conformational change. Stereo van der Waals diagrams before (Fig. 6b) and after binding MTT (Fig. 6c) reveal the closure of the active site and the formation of a well defined hydrophobic pocket on substrate/inhibitor binding.


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Fig. 6.   Ligand-induced conformational changes. a, r.m.s.d. plots of Calpha positions. Structural differences of monomer A between the adenine and MTT-bound structures (blue) and the FMA- and MTT-bound structures (red). The superimposition of the structures is as described under "Experimental Procedures." Residues 202-205 are disordered in the adenine-bound structure and are not present in the structure. The r.m.s.d. values for these residues are therefore zero. alpha -Helices and beta -sheets are labeled as defined in Lee et al. (18) and are shown as rectangles and arrows, respectively. Stereo van der Waals surface representation of the nucleosidase active site with adenine (b) and MTT (c) bound. Acidic residues are colored in red and basic residues are depicted in blue. The figures are drawn to the same scale to highlight the closure of the active site on inhibitor binding.

Adenine-binding Site Conformational Changes-- A comparison of the adenine and MTT-bound structures of MTA/AdoHcy nucleosidase reveals numerous backbone and side chain movements (Fig. 7). The largest movement is a loop to helix transition in residues 199-207. In the adenine-bound nucleosidase structure (Protein Data Bank code 1JYS), the electron density for residues 202-205 was either weak or missing. These residues were disordered in a solvent-exposed region of the enzyme. Upon binding of FMA or MTT, residues 200-211 form a N-terminal extension to the pre-existing alpha 6 helix (residues 206-229). This extended alpha 6 helix is not continuous as it has a ~45° kink at Leu211 (Fig. 7b). This conformational change results in a closure of the active site and the movement of residues 199-201 into the adenine-binding site where they make key hydrogen bonds to the enzyme, as described above. The amide nitrogen of Ala199 moves ~2 Å to make a hydrogen bond to Asp197 Odelta 2. The hydrogen bond together with one made between Asp197 Odelta 1 and Ser196 Ogamma are proposed to reposition the side chain of Asp197 closer to the N7 atom of the purine ring. The N-terminal extension of the alpha 6 helix and the additional hydrogen bonds made by Ala199 are likely important in closing the adenine-binding cavity. Thus, the hydrogen bond formed between the carbonyl oxygen of Ala199 and the amide nitrogen of Gly154 may serve to pull in residues 150-152 and hence shrink the adenine-binding cavity. Residues 150-152 shift ~1.2 Å, thus positioning Phe151 closer to the purine ring (Fig. 7c).


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Fig. 7.   Intrasubunit conformational changes upon inhibitor binding. a, ribbon diagram of the superimposed adenine (yellow) and MTT (blue) complexed MTA/AdoHcy nucleosidase monomers. b, ribbon diagram of the alpha 6 helix extension. c, stick diagram of the 150s loop shift. d, ribbon diagram of the helix alpha 1 shift. Figures were produced using Swiss-PDB Viewer (35) and POV-RAY.

The adenine molecule seen in the 1JYS structure is loosely bound as evident from the long hydrogen bond between the Asp197 and the N7 of adenine (~3.9 Å), and by the higher than average B-factors for the adenine (~55 Å2) versus the rest of the protein (33.7 Å2). The hydrogen bond between the N7 of the purine ring and the Odelta hydrogen of Asp197 in the FMA-complexed structure is now 2.7 Å (Fig. 4a). The N-terminal extension of the alpha 6 helix (residues 200-211) also allows Phe207 to move ~7.5 Å toward the 5'-alkylthio group to help complete the hydrophobic pocket. Phe151 and Phe207 make a herringbone interaction to Phe105 of the neighboring subunit.

Intramolecular 5'-Alkylthio-binding Site Conformational Changes-- A major intrasubunit conformational change involves a movement of residues 7-42. Residues Met9 and Glu12 in the alpha 1 helix undergo a movement into the active site upon ligand binding and this conformational change is propagated to residues in beta -strands, beta 2, beta 3, and beta 4 (Fig. 7d). Met9 moves toward the 5'-alkylthio-binding site. The side chain of Met9 packs against the 5'-methylthio group of MTT and helps create part of the hydrophobic pocket. Glu12 does not bind the substrate but hydrogen bonds a water molecule (WAT3) that is in close proximity to the C1' position of MTT (~3.5 Å). The conformational change of Glu12 creates hydrogen bonds to the amide hydrogen of Met9 and Ser76, and the Ogamma of Ser76. We propose that Glu12 is critical for the deprotonation of the nucleophilic water required for the hydrolysis of the N9-C1' bond (see later discussion).

The importance of the N-terminal residues of MTA/AdoHcy nucleosidase was previously analyzed with a truncation mutant of the first 8 residues (19). A high degree of sequence similarity exists with the first 8 residues of the human MTAP (VKIGIIGG) and the E. coli MTA/AdoHcy nucleosidase (MKIGIIGA). Cornell et al. (19) postulated that this region is involved in MTA or AdoHcy binding. Although the inhibitor-enzyme complexed structures show that residues 1-8 do not play a direct role in the binding of the substrate, elimination of these residues may affect the local structural stability of this region and therefore hinder the conformational changes required in the alpha 1 helix for catalysis, particularly residues Met9 and Glu12.

Intermolecular 5'-Alkylthio-binding Site Conformational Changes-- MTA/AdoHcy nucleosidase is thought to be a dimer based on the donation of residues in the 100s loop from a neighboring subunit. At the 5'-alkylthio tail, residues Val102, Phe105, Tyr107, and Pro113 were proposed to help make hydrophobic interactions to the 5'-methylthio or homocysteinyl group. The identity of these residues was determined using a superimposed model of MTA taken from the human MTAP structure (27). However, the modeled MTA molecule revealed that Tyr107, Pro113, and Val102 are ~8.7, 7.7, and 6.9 Å, respectively, away from the nearest atom in the 5'-methylthio group. This led us to suggest that an inter-subunit conformational change was probably needed to bring the residues into van der Waals contacts. The only residue modeled close enough to the 5' position of the substrate was Phe105 (4.4 Å).

The Calpha superimposition of the adenine and MTT-bound dimer reveals a conserved dimer interface (Fig. 8a). Upon further inspection, the 100s loop containing the donated residues was not found to undergo a major conformational change but rather a smaller loop translation by only some of the donated residues (Fig. 8b). Residues Val102 and Pro113 do not seem to undergo a conformational change but residues Phe105 and Tyr107 move ~1.5 Å closer to the methylthio group in both structures. This result is not surprising given that the 5'-methylthio group in the enzyme complexed with MTT is in a different conformation than the previously modeled MTA molecule. Residues Val102, Phe105, and Pro113 are actually in good van der Waals proximity to the methylthio tail of MTT. Even with the conformational changes seen in Tyr107, this residue is still too far removed (5.8 Å) to make van der Waals contacts with the methylthio group. However, as noted above Tyr107 Oeta could potentially hydrogen bond with the carboxyl moiety of the longer homocysteinyl group from AdoHcy.


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Fig. 8.   Intersubunit conformational changes upon inhibitor binding. a, ribbon diagram of the superimposed adenine and MTT complexed dimers. The dimers are superimposed as described under "Experimental Procedures" and are colored blue and green, respectively. b, a detailed view of the 100s loop involved in donating residues to the neighboring active site of the subunit. This figure was generated using Swiss-PDB Viewer (35) and POV-RAY.

Implications for Catalysis

MTA/AdoHcy nucleosidase is an inverting hydrolase that irreversibly cleaves the N9-C1' bond. The reaction catalyzed is similar to the N-ribohydrolase class of enzymes, which includes N-ribosyl hydrolases and N-ribosyltransferases, as well as the purine nucleoside phosphorylases. Given the similarities in structure and the similarities in the reactions catalyzed by the purine nucleoside phosphorylases, the nucleosidase has been proposed to share a similar two-step catalytic mechanism (18, 27, 31-34). The first step of the common catalytic mechanism requires a proton donation to the N7 atom of the adenine base. The proton donation allows a hydrogen bond to be formed between the N7 and enzyme. The purine base is electron withdrawing (electron pull) and is stabilized by a flow of electrons from the ribosyl O4' (electron push). This migration of electrons favors the elongation and the cleavage of the N9-C1' bond to form the oxocarbenium-like intermediate. The existence of the oxocarbenium intermediate is supported by kinetic isotope effect experiments (33). Following the formation of the oxocarbenium intermediate, an SN1-type attack by an activated nucleophilic water molecule occurs at the anomeric carbon to liberate the thioribose. Although the adenine-bound nucleosidase complex provided valuable structural insight into the catalytic mechanism, many specific details remain unanswered. In light of the inhibitor-enzyme complexes, we have re-examined the catalytic mechanism and have identified the catalytic base and nucleophilic water.

Identifying the Catalytic Base and Water-- Two water molecules (WAT2 and WAT3) were seen in the active site in proximity to the C1' atom of the modeled MTA molecule. WAT2 was originally proposed to be the nucleophilic water because of its proximity to the anomeric carbon (~4.2 Å). WAT2 was also hydrogen bonded to Glu172, which could act as a catalytic base. WAT3 was also in close proximity (~4.0 Å) but was not chosen because there was no neighboring catalytic base to activate the water. Analysis of the FMA- and MTT-bound structure reveals the displacement of WAT2 by the side chain of Arg193 and clearly identifies WAT3 as the catalytic water. In the MTT-bound structure, WAT3 is 3.6 Å from the anomeric carbon and 2.6 Å from Glu12 (Fig. 4b). In addition, WAT3 is in an excellent position for a rear attack on the ribosyl C1'. Glu12 is proposed to be the catalytic base because of its proximity to the nucleophilic water. Future site-directed mutagenesis experiments will confirm the role of Glu12 in catalysis.

Role of the 5' Thio Group-- In the purine nucleoside phosphorylases, sandwiching of the ribosyl O4' by two electron-rich groups, the negatively charged O4 phosphate and the 5' hydroxyl, is thought to push the flow of electrons from the ribosyl moiety to the purine base. When we superimposed MTA into the adenine-bound nucleosidase structure (18), our model identified the 5'-thio and the Ser76 Ogamma groups as being the electron-rich atoms in proximity to the O4' ribose. This observation seemed consistent with the biochemical data because modification of the sulfur to a less electronegative group such as selenium was known to decrease catalytic activity (19). However, the MTT complexed nucleosidase structure reveals that the 5'-thio group is in a different conformation than that seen in the human MTAP structure (Fig. 5) and likely does not play a role in the electron push. The two closest electron-rich atoms to the ribosyl O4' are Ser76 Ogamma (3.5 Å) and the nucleophilic water (3.7 Å). However, these groups do not sandwich the O4' ribose and instead are found on the same side of the ribose. In addition, Ser76 is not conserved in all nucleosidase species (Fig. 9) and the negatively charged nucleophilic water seems to be too far (3.7 Å) from the ribosyl O4' to facilitate an electron push. The current structures suggest that MTA/AdoHcy nucleosidase does not require the ribosyl O4' to be sandwiched between two electron-rich groups.


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Fig. 9.   Sequence alignment of various species of MTA/AdoHcy nucleosidase. Side chain and main chain atoms involved in substrate binding and catalysis are labeled with an asterisk and circle, respectively. Boxes highlighted in black show conserved sequences. The primary sequence alignment was performed in the program BioEdit using the ClustalW multiple alignment algorithm (37).

Although the 5' sulfur atom is likely not involved in the electron push and pull mechanism, it may have another role in catalysis. The FMA- and MTT-bound nucleosidases reveal structural differences that may provide an alternative explanation for the importance of the 5'-alkylthio sulfur. The FMA-bound structure reveals a different Glu12 conformation than the MTT-bound structure (Figs. 3 and 4). In the MTT-bound structure, Glu12 Oepsilon 1 makes hydrogen bond interactions to the amide nitrogen of Ala8 and Met9 and the nucleophilic water, and Glu12 Oepsilon 2 hydrogen bonds with the amide nitrogen and Ogamma of Ser76 and the nucleophilic water. A total of five hydrogen bonds are made to Glu12. The positioning of Glu12 in the MTT-bound structure is thought to be the catalytically active state with Glu12 in a position to activate the nucleophilic water. Unlike the MTT-complexed structure, Glu12 in the FMA-bound structure has a different conformation in each of the two active sites. In monomer A, Glu12 Oepsilon 1 is hydrogen bonded to Thr74 Ogamma 1, Ser218 Ogamma , and Ser219 N and Glu12 Oepsilon 2 is hydrogen bonded to Gly75 N, Thr74 Ogamma 1, and Ser218 Ogamma . Glu12 lacks the crucial hydrogen bonds to the nucleophilic water (WAT3) and Ser76. In monomer B, Glu12 hydrogen bonds to the nucleophilic water (WAT3), the amide nitrogen of Met9 and Glu12 but it lacks a hydrogen bond to the Ser76 Ogamma . Glu12 also makes three less hydrogen bonds and is farther away from the nucleophilic water (3.1 Å) than in the MTT-bound complex (2.6 Å). The binding of ligands with 5' hydroxyl groups seems to disrupt the proper positioning of the catalytic base, Glu12. This would account for the decrease in nucleosidase activity for 5'-hydroxyl containing nucleosides such as adenosine (19).

Substrate Specificity-- The inhibitor MTT (Ki = 0.75 µM) resembles the natural substrate MTA (Fig. 1) (Km = 0.5 µM) and has a very similar binding constant (19). Replacement of the methylthio group with a hydroxyl in FMA causes a ~20-fold decrease in the binding affinity (Ki = 10 µM) (19). The FMA- and MTT-inhibitor complexed structures reveal evidence to account for the lowered FMA binding affinity. FMA has a weaker binding constant because of the loss of van der Waals interactions made in the 5'-alkylthio hydrophobic pocket. In the MTT-complexed nucleosidase, the methylthio group makes van der Waals interactions to Met9, Ile50, Phe105, Phe151, Met173, and Phe207. In the FMA-bound nucleosidase, the 5'-hydroxyl has only van der Waals interactions to residues Ile50 and Met173. In addition, the unsatisfied hydrogen bond of the 5'-hydroxyl makes it further energetically unfavored. The decrease in FMA affinity may be partially offset by an additional hydrogen bond between Ser76 Ogamma and the N8 position of the purine.

Order of Product Release-- The order of substrate binding and product release in MTA/AdoHcy nucleosidase has not been experimentally characterized. The structure reveals that the adenine-binding site is deeply buried and the ribose- and 5'-alkylthio-binding sites are solvent exposed (Fig. 6c). This architecture suggests that the thioribose product is released first followed by the purine base. The purine base may not be released immediately as adenine is known to be a weak competitive inhibitor of E. coli MTA/AdoHcy nucleosidase (Ki = 300 µM) (19). In the adenine-complexed nucleosidase structure, the loosely bound adenine was salvaged from the cell and remained in the active site throughout purification (18). The residual adenine is likely only displaced when a new substrate molecule (MTA or AdoHcy) binds.

Conservation of Active Site Residues-- A sequence alignment of nucleosidases from 8 different organisms was performed. The residues involved in catalysis and substrate binding have varying degrees of conservation but their overall chemical nature is generally maintained (Fig. 9). Not surprisingly, the catalytic residues (Glu12 and Asp197) are invariant as are the residues involved in coordinating the catalytic water (Arg193 and Glu174) and orienting the catalytic acid (Ser196). The catalytic acid (Asp197) is also held in place by the main chain amide of Ala199. In H. influenzae Ala199 is replaced by a glycine. This substitution is not expected to perturb the local structure or affect the positioning of Asp197. Ile152 and Met173 also make main chain hydrogen bonds to the substrate. Whereas some variation is observed at position 152 with substitutions to leucine and valine, Met173 is strictly conserved in keeping with the additional role its side chain plays in stabilizing and orienting the ribose moiety of the substrate. With the exception of Ser76, the other adenine- and ribose-binding site residues that interact through their side chains are fairly well conserved. The hydrophobic nature of Phe151 and the 5'-alkylthio binding residues Met9, Ile50, and Phe207 are all maintained in the aligned nucleosidase species. The hydrophobic residues donated from the neighboring subunit (Val102, Phe105, Tyr107, and Pro113) are, however, more variable. Val102 maintains its hydrophobic nature with substitutions to alanine or leucine, but Phe105 and Tyr107 have been substituted in certain species with aspartate and histidine, respectively. It is unclear what role the aspartate may play in substrate binding. Although the replacement of tyrosine to histidine may look like a fairly drastic substitution, the proposed hydrogen bond between Tyr107 and the homocysteinyl tail of AdoHcy could potentially be maintained with a histidine. Pro113 is well conserved, with the exception of an alanine in S. pneumoniae.

    ACKNOWLEDGEMENTS

We thank Dr. G. David Smith for the program PROFIT v6.0 and help and insight into the analysis of the structures and Dr. Yuri D. Lobsanov for assistance at the Hospital for Sick Children x-ray diffraction facility. Fig. 1 was kindly prepared by Tom Rodinger.

    FOOTNOTES

* This work was supported in part by Canadian Institutes for Health Research Grant 43998 (to P. L. H.) and a doctoral research award (to J. E. L.). Station X8-C was supported by the United States Department of Energy and a Multiuser maintenance grant from the Canadian Institutes for Health Research and the Natural Science and Engineering Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1NC3 and 1NC1 for the formycin A and MTT complexes) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

** Supported by the United States Veterans Affairs Medical Research Program.

§§ To whom correspondence should be addressed. Tel.: 416-813-5378; Fax: 416-813-5022; E-mail: howell@sickkids.ca.

Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M210836200

    ABBREVIATIONS

The abbreviations used are: MTA, 5'-methylthioadenosine; AdoHcy, S-adenosylhomocysteine; FMA, formycin A; MTT, 5'-methylthiotubercidin; MTAP, 5'-methylthioadenosine phosphorylase; r.m.s.d., root mean square deviation.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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