J Biol Chem, Vol. 274, Issue 30, 21114-21120, July 23, 1999

Nucleoside Hydrolase from Leishmania major
CLONING, EXPRESSION, CATALYTIC PROPERTIES, TRANSITION STATE INHIBITORS, AND THE 2.5-Å CRYSTAL STRUCTURE^*

Wuxian Shi, Vern L. Schramm, and Steven C. Almo

From the Albert Einstein College of Medicine, Department of Biochemistry, Bronx, New York 10461

	ABSTRACT

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Protozoan parasites lack the pathway of the de novo synthesis of purines and depend on host-derived nucleosides and nucleotides to salvage purines for DNA and RNA synthesis. Nucleoside hydrolase is a central enzyme in the purine salvage pathway and represents a prime target for the development of anti-parasitic drugs. The full-length cDNA for nucleoside hydrolase from Leishmania major was cloned and sequence analysis revealed that the L. major nucleoside hydrolase shares 78% sequence identity with the nonspecific nucleoside hydrolase from Crithidia fasciculata. The L. major enzyme was overexpressed in Escherichia coli and purified to over 95% homogeneity. The L. major nucleoside hydrolase was identified as a nonspecific nucleoside hydrolase since it demonstrates the characteristics: 1) efficient utilization of p-nitrophenyl -D-ribofuranoside as a substrate; 2) recognition of both inosine and uridine nucleosides as favored substrates; and 3) significant activity with all of the naturally occurring purine and pyrimidine nucleosides. The crystal structure of the L. major nucleoside hydrolase revealed a bound Ca²⁺ ion in the active site with five oxygen ligands from Asp-10, Asp-15 (bidentate), Thr-126 (carbonyl), and Asp-241. The structure is similar to the C. fasciculata IU-nucleoside hydrolase apoenzyme. Despite the similarities, the catalytic specificities differ substantially. Relative values of k_cat for the L. major enzyme with inosine, adenosine, guanosine, uridine, and cytidine as substrates are 100, 0.5, 0.5, 27 and 0.3; while those for the enzyme from C. fasciculata are 100, 15, 14, 510, and 36 for the same substrates. Iminoribitol analogues of the transition state are nanomolar inhibitors. The results provide new information for purine and pyrimidine salvage pathways in Leishmania.

	INTRODUCTION

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Leishmania species are protozoan parasites transmitted by blood-sucking female phlebotomine sandflies. They infect humans primarily in the Middle East, central Asia, and Africa, but are found on all continents except Australia (1). Current chemotherapeuric treatments are also toxic to the host. Most of the protozoan parasites lack the pathway for de novo purine synthesis and depend on purine salvage from the host for DNA and RNA synthesis (2). Several enzymes have been implicated in the purine salvage pathway, including a cell-surface associated enzyme, 3'-nucleotidase/nuclease, that is proposed to generate nucleosides. The nucleosides are further metabolized by nucleoside hydrolases, a family of enzymes which hydrolyze the N-glycosidic bond of purine and pyrimidine ribosides to yield ribose and the bases (3, 4). Phosphoribosyl transferases are proposed to form the respective nucleotides by reaction of the bases with 5-phosphoribosyl-1-pyrophosphate (5). Nucleoside hydrolases and phosphoribosyl transferases have been characterized from several sources, and the identification of inhibitors for these enzymes might be of use in parasite infections (6-10). Structures for the nucleoside hydrolase from Crithidia fasciculata were reported recently, however, there is no structural information on nonspecific nucleoside hydrolases from protozoan parasites with human hosts. The purpose of this study was to evaluate the properties of nucleoside hydrolase from Leishmania relative to those from Crithidia and Trypanosoma (11, 13).

Cell-free extracts from a variety of protozoan parasites indicate the existence of several nucleoside hydrolase isozymes with differing substrate specificities. The most abundant nucleoside hydrolase in C. fasciculata is IU¹-nucleoside hydrolase, a nonspecific enzyme that catalyzes efficient hydrolysis of both purine and pyrimidine nucleosides (11). A second nucleoside hydrolase from C. fasciculata, GI-nucleoside hydrolase, prefers purine nucleosides as substrates (12). In addition to the two nucleoside hydrolases from C. fasciculata, a purine-preferring nucleoside hydrolase from the bloodstream form of Trypanosoma brucei brucei has been characterized and named IAG-nucleoside hydrolase since the enzyme prefers inosine, adenosine, and guanosine as substrates (13).

The IU-nucleoside hydrolase from C. fasciculata is the most extensively studied enzyme in protozoan purine salvage pathways. It has been cloned and expressed in Escherichia coli and mutagenesis studies have identified His-241 as a catalytic site residue that acts as a general acid for leaving-group activation (14). The transition state structure of IU-nucleoside hydrolase, determined by kinetic isotope effect measurements, has been shown to have characteristics of an oxocarbenium ion (15). The three-dimensional structures of unliganded IU-nucleoside hydrolase and the enzyme complexed with p-aminophenyl-(1S)-iminoribitol, a transition state inhibitor, have been solved by x-ray crystallography to 2.5- and 2.3-Å resolution, respectively (7, 16-18).

The sequence alignment search using the amino acid sequence of the IU-nucleoside hydrolase from C. fasciculata found an est-cDNA clone of unknown function from Leishmania major. High sequence identity was established in the N-terminal 48 residues of deduced amino acid sequence (14). We report here the cloning of the full-length nucleoside hydrolase from L. major, determination of its primary amino acid sequence, its expression in E. coli, a purification scheme, analysis of substrate specificity, several nanomolar transition state inhibitors, and the 2.5-Å x-ray structure of the enzyme.

	MATERIALS AND METHODS

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Cloning and Sequencing-- cDNA clone LM247 phage stock was generously provided by Dr. J. W. Ajioka from the Laboratory for Parasite Genome Analysis at Cambridge University. Lambda phage lysates were prepared from LB plates with dense plaques using bacterial host strain XL1-blue (Novagen Inc.). Phage DNA was isolated using a phage purification kit (Novagen Inc.) and was used as a template for PCR reactions. A 5'-oligonucleotide primer 5'-GCATTTGCGAGCCATGCCG-3' was designed based on the partial sequence on the 5'-end of the LM247 clone that shows significant sequence identity to the gene sequence of IU-nucleoside hydrolase from C. fasciculata (14). The 5' primer was used with a 3'-oligonucleotide primer 5'-TAATACGACTCACTATAGCG-3' corresponding to the T7 promoter, which is located on the vector  ZAPII of the LM247 clone. A PCR product of approximately 1.0 kilobases was ligated into the TA cloning vector (Invitrogen Inc.). The full sequence of the PCR product showed an 78% amino acid sequence identity to the protein sequence of the IU-nucleoside hydrolase from C. fasciculata.

Expression-- Primers 5'-CATATGCCGCGCAAGATTATTCTC-3' and 5'-GGATCCTCACTGAGGATCGCCGAT-3' were based on the nucleotide sequence of the 5'- and 3'-regions of the sequenced PCR product and were designed to include restriction sites NdeI and BamHI, respectively. A stop codon, TGA, was also incorporated into the 3'-primer. These were used to amplify the full-length gene by PCR using LM247 Lambda phage DNA as template. The PCR product was purified from a 1% agarose gel using the Qiaquick gel extraction kit (QIAGEN Inc.). The cDNA was ligated into the expression vector PMW172 and the construct was transformed into E. coli strain BL21(DE3) for protein expression. Analysis by SDS-polyacrylamide gel electrophoresis indicated overexpression of a protein with molecular mass of approximately 34,000 daltons without isopropyl-1-thio--D-galactopyranoside induction. The crude cell extracts gave nucleoside hydrolase activity with both inosine and p-nitrophenyl -D-ribofuranoside as substrates.

Enzyme Purification-- The 0.95-kilobase open reading frame for nucleoside hydrolase in the PMW172 expression vector was expressed in BL21(DE3) E. coli cells (Novagen Inc.). Cells were grown in LB medium containing 50 µg/ml ampicillin. Cell cultures were grown continuously in shaken flasks for 7 h at 37 °C and harvested by centrifugation. The cells were washed with and resuspended in an equal volume 20 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, 1 mM EDTA, 15 µM phenylmethylsulfonyl fluoride, and 50 mM NaCl. The cells were frozen and stored at 70 °C.

Frozen cells were thawed in warm water and disrupted with 4 cycles of French press treatment. The cell lysate was centrifuged at 15,000 rpm for 80 min. Neomycin sulfate (10% w/v in H₂O) was added slowly with vigorous stirring to a final concentration of 1% (w/v). After centrifugation, solid ammonium sulfate was added to the supernatant to a final concentration of 40%. The pellet was collected by centrifugation, resuspended in a minimum volume of 20 mM triethanolamine, pH 7.8, containing 15 µM phenylmethylsulfonyl fluoride (buffer A), and dialyzed overnight at 4 °C against the same buffer. The dialysate was applied to a FastQ anion exchange chromatography column equilibrated with buffer A. The column was washed with 1.5 column volumes of buffer A and eluted with 4 column volumes of buffered 0-0.3 M NaCl in a linear gradient. The nucleoside hydrolase was eluted between 2.5 and 3.0 column volumes. The fractions containing the nucleoside hydrolase were pooled, concentrated, and loaded on a Superdex 200 chromatography column equilibrated with 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1 mM EDTA and eluted with the same buffer. The fractions containing nucleoside hydrolase activity were pooled, dialyzed against 20 mM triethanolamine, pH 7.3, and concentrated by ultrafiltration. The protein was rapidly frozen and stored at 70 °C.

Crystallization-- Recombinant nucleoside hydrolase from L. major was crystallized using hanging-drop vapor diffusion at 18 °C. The protein was crystallized in the presence of hypoxanthine, which is a product of the enzyme and also inhibits the enzyme at high concentration. Saturated hypoxanthine (6 µl) was air dried on a glass coverslip. On top of the dried hypoxanthine, 3 µl of 13-15 mg/ml protein was mixed with an equal volume of mother liquid containing 100 mM MES, pH 6.5, and 16% polyethylene glycol 4000, and was equilibrated against 1.0 ml of the mother liquid in the well. Crystals appeared in 3-5 days in the presence of some amorphous precipitate. Crystals grew in clusters, but single crystals could be separated from the clusters. Diffraction from these crystals is consistent with the monoclinic space group P2₁ with a = 81.8 Å, b = 79.2 Å, c = 109.8 Å, and  = 91.6^o. The crystals contain a tetramer in the asymmetric unit with a V_m = 2.54 Å³/Da and a calculated solvent content of 56%.

Data collection and Processing-- X-ray diffraction data were collected at room temperature from a single crystal of nucleoside hydrolase using a Siemens X-1000 area detector coupled to a Rigaku rotating anode x-ray generator operating at 50 KV and 80 mA. Radiation from the copper anode was focused through Cross-coupled Gobel mirrors (Bruker AXS) and a 0.3-mm collimator. Indexing and integration of the data frames were carried out using program XDS (19) and the data was scaled using SCALEPACK from the DENZO package (20). The data set used for the molecular replacement and structural refinement had an overall completeness of 84% to 2.5-Å resolution with an R_sym of 7.2%. Data collection statistics are shown in Table I.

Molecular Replacement and Structural Refinement-- The structure of nucleoside hydrolase from L. major was solved by molecular replacement using the AmoRe software package implemented in CCP4 (21). Coordinates of the IU-nucleoside hydrolase dimer from C. fasciculata were used as the search model with all side chains that are different from L. major nucleoside hydrolase trimmed to alanine (7). Temperature factors of all atoms were reset to 20.0 Å². Two solutions, corresponding to the orientations and locations of the two homodimers in the asymmetric unit, were generated using 8.0-4.0 Å data.

The tetrameric structure generated by molecular replacement was subjected to rigid-body refinement using XPLOR (22). The R_cryst dropped from 41.7 to 32.6% and R_free was also reduced from 39.5% to 31.5 after 40 steps of rigid-body refinement using 8.0-4.0 Å data. Program O (23) was used to view the structure and build the missing side chains into the electron densities in the 2 F_o  F_c  map. Strict noncrystallographic symmetry constraints were applied in the initial cycle of refinement and relaxed in the subsequent cycles. The final structure contains residues 2-313 in all four subunits, one Ca²⁺ for each subunit and a total of 100 water molecules in the tetramer with R_cryst of 20.3% and R_free of 25.5%. The model shows good geometry with 85.3% of the residues in the most favored region, 13.6% in the additionally allowed region, and 1.1% in the generously allowed region by PROCHECK (24). Statistics for the refinement are summarized in Table I.

Substrate Activity Assay-- Nucleoside hydrolase was assayed at room temperature or at 30 °C in 50 mM Hepes, pH 8.0. Hydrolysis of inosine to hypoxanthine and ribose was followed by continuous reading of optical absorbance at 280 nm (11). Hydrolysis of p-nitrophenyl -D-ribofuranoside was followed by release of the p-nitrophenylate anion which has strong absorbance at 400 nm with extinction coefficient of 14,600 M¹ cm¹ under the assay condition (25). The hydrolysis of guanosine, adenosine, cytidine, and uridine was monitored by release of reducing sugar at room temperature (11). The inhibition constants for inhibitors were measured at a fixed concentration of p-nitrophenyl -D-ribofuranoside and variable concentrations of inhibitor. The results were fit to the equation: v = V_mA/[K_m(1 + I/K_i) + A], where v = the initial rate of product formation, V_m = maximum catalytic rate, A = substrate concentration, K_m = Michaelis constant, and K_i = the dissociation constant for the enzyme-inhibitor complex. This analysis makes the assumption that A and I (the inhibitor) compete for the catalytic site.

	RESULTS AND DISCUSSION

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Sequence Comparison-- The amino acid sequence of the nucleoside hydrolase from L. major contains a total of 315 residues including the N-terminal Met (Table II). Sequence alignment was performed with the IU-nucleoside hydrolase from C. fasciculata and the IAG-nucleoside hydrolase from T. brucei brucei (14, 25). The L. major nucleoside hydrolase shares 78% sequence identity with the IU-nucleoside hydrolase but only 20% sequence identity with the IAG-nucleoside hydrolase. The IU-nucleoside hydrolase from C. fasciculata requires the presence of all three hydroxyl groups, (2'-, 3'-, and 5'-hydroxyls), in the nucleosides to ensure efficient substrate binding and catalysis (11). Residues involved in hydrogen bonding of the three hydroxyls in the C. fasciculata enzyme include Asp-10, Asp-14, Asp-15, Asn-39, and Asp-242 (Asp-241 in L. major because of one deletion in the amino acid sequence) for the 2'- and 3'-hydroxyls and Asn-160, Glu-166, and Asn-168 for the 5'-hydroxyl (7). All these residues involved in substrate recognition are conserved in the L. major nucleoside hydrolase (Table II). In the IAG-nucleoside hydrolase, the only residues in contact with ribosyl hydroxyls that are not conserved are Asn-39 and Asn-160. In IAG-NH, Asn-39 is replaced by an aspartate which has a similar hydrogen bonding ability, and Asn-160 is replaced by Thr near a 6-amino acid insertion (Table II). The insertion of amino acids GNVFLP in IAG-NH prior to the position corresponding to Asn-160 in the L. major enzyme is likely to be involved in the ability of the IAG-NH to hydrolyze deoxynucleosides more efficiently than the nonspecific nucleoside hydrolases (11, 13).

His-241 in the IU-NH from C. fasciculata has been proposed to be involved in leaving group activation. Mutation of H241A in the IU-NH resulted in over 2000-fold loss of activity in inosine hydrolysis with no loss in the rate of hydrolysis of p-nitrophenyl -D-ribofuranoside, a substrate that is not activated by leaving group protonation (14, 26). This His residue (242) is conserved in the L. major nucleoside hydrolase. The IAG-nucleoside hydrolase from T. brucei brucei has a Trp in this position which is likely to be involved in its leaving group specificity for purines, possibly as a result of base stacking with Trp. At present there is no structural information for the IAG-nucleoside hydrolase.

Substrate Specificity-- The extensive amino acid sequence similarity between nucleoside hydrolases from C. fasciculata and L. major suggested closely related catalytic properties. The p-nitrophenyl -D-ribofuranoside has been shown to be a good substrate for the nonspecific nucleoside hydrolase but a poor one for purine-specific nucleoside hydrolases including the IAG-NH from T. brucei brucei (13). This discrimination arises from differences in the catalytic mechanism for transition state formation. Purine-specific nucleoside hydrolases lower the energy of activation to achieve the transition state by applying similar energies to both leaving group activation (protonation or hydrogen bonding) and ribosyl activation (forming a ribooxocarbenium ion). Nonspecific nucleoside hydrolases typified by the IU-NH from C. fasciculata invest most of their catalytic energy into ribooxocarbenium formation. The substrate p-nitrophenyl -D-ribofuranoside cannot be protonated on the leaving group, but is susceptible to fascile decomposition when the ribosyl group is converted to the oxocarbenium ion. The nucleoside hydrolase from L. major demonstrates the characteristics expected for nonspecific nucleoside hydrolases, including, 1) efficient utilization of p-nitrophenyl -D-ribofuranoside as a substrate; 2) recognition of both inosine and uridine nucleosides as favored substrates; and 3) some catalytic activity with all of the naturally occurring purine and pyrimidine nucleosides (Table III). Despite these similarities between the C. fasciculata and L. major enzymes, differences exist in catalytic specificity and efficiency. The k_cat values for inosine of 28 s¹ for the C. fasciculata and 119 s¹ for the L. major establish a 4.25-fold increased catalytic turnover for the best purine substrate. The specificity for purines and pyrimidines also differs significantly. Catalytic turnover ratios for uridine/inosine are 5.1 for the C. fasciculata enzyme but only 0.27 for that from L. major. Both the k_cat and relative rates of hydrolysis of adenosine and guanosine are slower for the L. major enzyme (Table IV). These properties indicate that the L. major enzyme has evolved to catalyze an increased turnover of inosine, with a loss of catalytic efficiency for other nucleosides. Hypoxanthine is thought to be the major precursor for purine salvage in L. major, and the substrate specificity of this enzyme is well suited to the role (1, 2). Metabolic efficiency in a salvage pathway benefits from enzymes with k_cat/K_m values near diffusion control, approximately 10⁸ M¹ s¹ for the nucleoside hydrolases. The nonspecific nucleoside hydrolase described here is approximately 2 orders of magnitude from catalytic perfection because of a high K_m value. It is possible that inosine transport by the protozoa provides intracellular concentrations above the low micromolar levels found in blood.

Transition-state Inhibitors for L. major Nucleoside Hydrolase-- Iminoribitols with 1-S substitutions are transition state inhibitors for the IU-NH from C. fasciculata (6). Inhibitors containing 9-deazadenosine (immucillin A) or 4-amino-5-carbonylamino-3-pyrrolyl (immucillin ACAP) substituents of iminoribitol have been shown to inhibit both C. fasciculata and T. brucei brucei nucleoside hydrolases with nanomolar inhibition constants.² These analogues of the nucleoside hydrolase transition state gave inhibition constants of 6.5 and 15 nM for the nucleoside hydrolase from L. major (Table V). These dissociation constants vary less than 2-fold from those of C. fasciculata nucleoside hydrolase but differ more from the K_i values obtained with the enzyme from T. brucei brucei. Inosine is the best substrate for nucleoside hydrolase from L. major with a K_m of 445 µM. The inhibitors of Table V are powerful inhibitors for this enzyme with immucillin A binding 29,700 times more tightly than the K_m for inosine, while immucillin ACAP gives a K_m/K_i ratio of 68,460. These inhibitors would compete very favorably for the catalytic site of nucleoside hydrolase, even in the presence of nanomolar inhibitor and micromolar substrate.

Overall Fold of the L. major Nucleoside Hydrolase-- Nucleoside hydrolase from L. major is folded into a single domain structure containing 12 -helices and 10 -strands (Fig. 1). The core of the structure consists of a 7-stranded parallel -sheet (3, 63-65; 2, 28-35; 1, 3-9; 4, 121-125; 5, 148-152; 6, 186-189; and 10, 288-290) and two -helices (1, 13-24 and 11, 242-249). Five -helices (3, 105-115; 4, 130-138; 5, 141-145; 7, 173-180; and 6, 167-170) stack above the central -sheet and three -helices (10, 213-228; 12, 296-310; and 9, 201-210) sit beneath the two helices (1, 11) in the core. Two -helices (2, 42-55 and 8, 191-194) are located at the either side of the core. Two 2-stranded antiparallel -sheets (7, 254-257 and 10, 291-294; 8, 261-263 and 9, 274-276) are oriented perpendicular to each other and protecting the core structure from the right side. A bound Ca²⁺ ion is located in the active site cleft formed by 2, 1, 4, 5, 1, and 11 in the core. The enzyme was co-crystallized with saturating hypoxanthine. Although the addition of hypoxanthine improved the crystal morphology, no hypoxanthine molecules were found either in the active site or other part of the protein structure. The long loop connecting 3 and 3 is projecting outwards and is very flexible. Part of this loop (residues 76-83) was modeled as polyalanine because of the poor side chain densities in the electron density maps.

View larger version (55K): [in this window] [in a new window]   Fig. 1.   Stereoview of the monomer of L. major nucleoside hydrolase (a) with a view of the bound Ca2+ (green). The subunit packing (b) demonstrates the novel interfaces of the tetramer with the locations of the catalytic sites indicated with Ca2+. Figs. 1-3 were generated using SETOR (27).

Quaternary Structure-- L. major nucleoside hydrolase forms a homotetramer that is related by a 222 point symmetry in the asymmetric unit. The four molecules are nearly identical because of the noncrystallographic symmetry restraints applied in the refinement. The packing of the tetrameric structure is rather loose. The tightest interface (A/B or C/D) in the tetramer buries a total of 1957 Å² surface area, which represents 7.3% of the total solvent accessible surface area for each molecule. The A/B interface includes the 8/9 (263-282) region and the connection between 5 and 6 (155-165). The interactions are mostly hydrophobic van der Waals contributed by Leu-269, Val-275 from both subunits and two hydrogen bond pairs between side chain of Glu-264 and backbone N of Leu-269, and between side chains of His-157. The other two interfaces (A/D or B/C, A/C or B/D) bury a total of 1685 and 82 Å² surface area, respectively.

Ca²⁺-binding Site-- The crystal structures of apo and complexed IU-nucleoside hydrolase from C. fasciculata have revealed a Ca²⁺ ion bound at the active site (7, 18). Elemental analysis confirmed that the Ca²⁺ binds to the IU-nucleoside hydrolase with 1:1 molar ratio (7). In the L. major nucleoside hydrolase crystal structure, a single Ca²⁺ ion is located in each of the molecules in the tetramer and the electron density in the 2 F_o  F_c  maps indicates that the Ca²⁺ ion is fully occupied in the active site. Crystal structures of Ca²⁺ complexes indicate that Ca²⁺ usually binds to oxygen atoms in chelating complexes and the preferred coordination numbers range from 6 to 8 (28). In the L. major nucleoside hydrolase, however, only five oxygen ligands are found chelating the Ca²⁺. The remaining chelation sites are open to solvent contact and it is likely that one or more of these additional sites are in exchange with water. In the structure of IU-NH from C. fasciculata, the sixth chelate position was occupied by an ordered water, but no ordered water is seen here. The distances for the coordination are 2.6 Å to the OD1 of Asp-10, 2.8 and 2.9 Å, respectively, to the OD2 and OD1 of Asp-15, 2.7 Å to the OD2 of Asp-241, and 2.5 Å to the carbonyl O of Thr-126 (Fig. 2). The Ca²⁺ is located near the bottom of the active site cleft and the side chains of these residues point up to chelate the Ca²⁺. In the C. fasciculata IU-nucleoside hydrolase apoenzyme structure, the coordination to Ca²⁺ from protein atoms is very similar, but a water molecule is located above the Ca²⁺ to provide the sixth ligand. In the C. fasciculata structure with bound inhibitor, the 2'- and 3'-hydroxyls from the bound transition state inhibitor provide two more ligands to the Ca²⁺. The mechanistic role of the Ca²⁺ is to orient substrate in the active site and to position the water molecule for attack at the C1'-anomeric carbon following the formation of the ribooxocarbenium ion (7). The amino acid residues that make contact to the Ca²⁺ are completely conserved in all three of the well characterized nucleoside hydrolases (Table II).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Stereoview of the contacts between L. major nucleoside hydrolase with the Ca2+ (blue). The distances are in angstroms.

Structural Comparison of Nucleoside Hydrolases-- The L. major nucleoside hydrolase is structurally similar to the C. fasciculata IU-nucleoside hydrolase (Fig. 3). This is expected from the 78% amino acid sequence identity and conservation of sequences in the catalytic site. The overlap of C traces of the two structures reveals differences in the two flexible loop regions. These are the long loop connecting 3 and 3 (76-83), and the loop immediately after 10 (230-235). The root mean square deviation between all C atoms excluding the two flexible loop regions is 0.4 Å between the L. major enzyme and the C. fasciculata apoenzyme, and 0.8 Å between the L. major enzyme and the C. fasciculata complex structure. In the C. fasciculata complex structure, the two flexible loops moved closer to the active site of the enzyme upon binding of a transition state inhibitor (7).

View larger version (62K): [in this window] [in a new window]   Fig. 3.   Stereoview comparison of the L. major nucleoside hydrolase with the C. fasciculata nucleoside hydrolase apoenzyme (a) and the enzyme complexed with p-aminophenyl-(1S)-iminoribitol, a transition state inhibitor (b). Most of the differences occur in the long loop connecting 3 and 3 (Loop I) and the loop immediately after 10 (Loop II).

Conclusions-- L. major is the causative parasite for one of the significant world health infectious diseases. This report is the first structural description of a nucleoside hydrolase from the organism. Although the enzyme is established as a member of the nonspecific nucleoside hydrolases, it has a unique substrate specificity with strong preference for inosine. Its mechanism includes a tightly bound catalytic site Ca²⁺. Two inhibitors are described that bind >10⁵-fold tighter than the K_m for substrate. This information may be useful in the design of anti-Leishmania agents.

	ACKNOWLEDGEMENTS

We thank Drs. Peter Tyler and Richard Furneaux of the Carbohydrate Chemistry Team, Industrial Research Ltd., for supplying the iminoribitol inhibitors of Table V. We thank Dr. Robert Miles for determination of K_i values for the L. major nucleoside hydrolase.

	FOOTNOTES

^* This work was supported by Research Grant GM41916 from the National Institutes of Health.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.

To whom correspondence should be addressed. E-mail: almo@ aecom.yu.edu.

² R. W. Miles, P. C. Tyler, G. B. Evans, R. H. Furneaux, D. W. Parkin, and V. L. Schramm, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: IU, inosine-uridine; GI, guanosine-inosine; IAG, inosine, adenosine, guanosine; PCR, polymerase chain reaction; MES, 4-morpholineethanesulfonic acid.

	REFERENCES

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