Crystal structure of purine nucleoside phosphorylase from Thermus thermophilus

The purine nucleoside phosphorylase from Thermus thermophilus crystallized in space group P4(3)2(1)2 with the unit cell dimensions a = 131.9 A and c = 169.9 A and one biologically active hexamer in the asymmetric unit. The structure was solved by the molecular replacement method and refined at a 1.9A resolution to an r(free) value of 20.8%. The crystals of the binary complex with sulfate ion and ternary complexes with sulfate and adenosine or guanosine were also prepared and their crystal structures were refined at 2.1A, 2.4A and 2.4A, respectively. The overall structure of the T.thermophilus enzyme is similar to the structures of hexameric enzymes from Escherichia coli and Sulfolobus solfataricus, but significant differences are observed in the purine base recognition site. A base recognizing aspartic acid, which is conserved among the hexameric purine nucleoside phosphorylases, is Asn204 in the T.thermophilus enzyme, which is reminiscent of the base recognizing asparagine in trimeric purine nucleoside phosphorylases. Isothermal titration calorimetry measurements indicate that both adenosine and guanosine bind the enzyme with nearly similar affinity. However, the functional assays show that as in trimeric PNPs, only the guanosine is a true substrate of the T.thermophilus enzyme. In the case of adenosine recognition, the Asn204 forms hydrogen bonds with N6 and N7 of the base. While in the case of guanosine recognition, the Asn204 is slightly shifted together with the beta(9)alpha(7) loop and predisposed to hydrogen bond formation with O6 of the base in the transition state. The obtained experimental data suggest that the catalytic properties of the T.thermophilus enzyme are reminiscent of the trimeric rather than hexameric purine nucleoside phosphorylases.     

Synthesis of 6-(2-thienyl)purine nucleoside derivatives that form unnatural base pairs with pyridin-2-one nucleosides

Unnatural bases, 2-amino-6-(2-thienyl)purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace the previously developed purine analogue, 2-amino-6-(N,N-dimethylamino)purine, which specifically pairs with pyridin-2-one. These nucleoside derivatives were synthesized via the 6-substitution of 6-iodopurine nucleosides with tributylstannylthiophene or tributylstannylfuran. As compared with 2-amino-6-(N,N-dimethylamino)purine, 2-amino-6-(2-thienyl)purine reduced the interference in the stacking interactions with the neighboring bases in a DNA duplex and improved the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite the unnatural base.     

Synthesis of 6-(2-thienyl)purine nucleoside derivatives toward the expansion of the genetic code.

Unnatural bases specifically pairing with pyridin-2-one, 2-amino-6-(2-thienyl) purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace 2-amino-6-(N,N-dimethylamino)purine. It was expected that these novel purine analogues, as compared with 2-amino-6-(N,N-dimethylamino)purine, might reduce the interference in the stacking interactions with the neighboring bases in a duplex and improve the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite these unnatural bases. The syntheses of these nucleoside derivatives and the DNA fragments were examined.     

Inhibition of RNA polymerase and formyltetrahydrofolate synthetase activity by 6-chloro-8-aza-9-cyclopentylpurine. Structure-activity relationships

The structural requirements for inhibition of bacterial RNA polymerase and rabbit liver formyltetrahydrofolate synthetase activity by a series of purine nucleoside analogs related to 6-chloro-8-aza-9-cyclopentylpurine (689) were investigated. To achieve an inhibitory effect, preincubation of the enzyme preparations with the purine analogs, prior to assay of enzyme activity, was required. The greatest inhibition was produced by analogs containing all three alterations of the purine nucleoside structure: the 6-halo, 8-aza, and 9-cyclopentyl groups. It is suggested that 689 inhibits the activity of enzymes involved in nucleic acid synthesis by a site-directed alkylation.     

Specificity of nucleoside transport in Neurospora crassa.

The specificity of nucleoside uptake in germinating conidia of Neurospora crassa was investigated by examining the kinetics of [2-14C]uridine and [8-14C]-adenosine uptake in the wild-type, ad-8, and ud-1 pyr-1 strains. The results obtained strongly indicate that nucleoside transport in N. crassa is mediated solely by a general transport system which accepts both purine and pyrimidine nucleosides. Studies directed at characterizing the specificity of the transport system indicate that general structural features of the nucleoside which enhance its efficiency in binding to the transport system include: (i) a purine or pyrimidine as the heterocyclic ring, (ii) an unfunctionalized ribose or 2'-deoxyribose as the sugar unit, (iii) a beta-configuration about the anomeric carbon, (iv) the absence of substituents at C8 in the purine series and at C5 and C6 in the pyrimidine series, (v) the presence of a C5-C6 double bond in the pyrimidine series, and (vi) the absence of a charge on the heterocyclic ring.     

An enzymatic transglycosylation of purine bases

An enzymatic transglycosylation of purine heterocyclic bases employing readily available natural nucleosides or sugar-modified nucleosides as donors of the pentofuranose fragment and recombinant nucleoside phosphorylases as biocatalysts has been investigated. An efficient enzymatic method is suggested for the synthesis of purine nucleosides containing diverse substituents at the C6 and C2 carbon atoms. The glycosylation of N(6)-benzoyladenine and N(2)-acetylguanine and its O(6)-derivatives is not accompanied by deacylation of bases.     

Colorimetric Determination of α-Amino Nitrogen in Urine and Plasma with Ninhydrin Reaction

Bacillus stearothermophilus TH 6–2 has two kinds of purine nucleoside phosphorylases (Pu-NPase I and Pu-NPase II). The Pu-NPase I is a functional homolog of eukaryotic purine nucleoside phosphorylases that can catalyze the phosphorolysis of inosine and guanosine, but not adenosine, the primary substrate of Pu-NPase II. The Pu-NPase I gene of TH 6–2 has been cloned, sequenced, and expressed in E. coli. The gene corresponded to an open reading frame of 822 nucleotides that translates into a putative 274-amino acid protein with a molecular weight of 29,637. The deduced amino terminus sequence completely coincided with that found for the purified enzyme. The cloned gene was overexpressed in E. coli by using the trc promoter to produce an active enzyme in large quantities. The amino acid sequence of Pu-NPase I shared 50% similarity with those of human and mouse purine nucleoside phosphorylases.     

Ethenoguanines Undergo Glycosylation by Nucleoside 2′-Deoxyribosyltransferases at Non-Natural Sites

Deoxyribosyl transferases and functionally related purine nucleoside phosphorylases are used extensively for synthesis of non-natural deoxynucleosides as pharmaceuticals or standards for characterizing and quantitating DNA adducts. Hence exploring the conformational tolerance of the active sites of these enzymes is of considerable practical interest. We have determined the crystal structure at 2.1 Å resolution of Lactobacillus helveticus purine deoxyribosyl transferase (PDT) with the tricyclic purine 8,9-dihydro-9-oxoimidazo[2,1-b]purine (N ²,3-ethenoguanine) at the active site. The active site electron density map was compatible with four orientations, two consistent with sites for deoxyribosylation and two appearing to be unproductive. In accord with the crystal structure, Lactobacillus helveticus PDT glycosylates the 8,9-dihydro-9-oxoimidazo[2,1-b]purine at N7 and N1, with a marked preference for N7. The activity of Lactobacillus helveticus PDT was compared with that of the nucleoside 2′-deoxyribosyltransferase enzymes (DRT Type II) from Lactobacillus leichmannii and Lactobacillus fermentum, which were somewhat more effective in the deoxyribosylation than Lactobacillus helveticus PDT, glycosylating the substrate with product profiles dependent on the pH of the incubation. The purine nucleoside phosphorylase of Escherichia coli, also commonly used in ribosylation of non-natural bases, was an order of magnitude less efficient than the transferase enzymes. Modeling based on published active-site structures as templates suggests that in all cases, an active site Phe is critical in orienting the molecular plane of the purine derivative. Adventitious hydrogen bonding with additional active site residues appears to result in presentation of multiple nucleophilic sites on the periphery of the acceptor base for ribosylation to give a distribution of nucleosides. Chemical glycosylation of O ⁹-benzylated 8,9-dihydro-9-oxoimidazo[2,1-b]purine also resulted in N7 and N1 ribosylation. Absent from the enzymatic and chemical glycosylations is the natural pattern of N3 ribosylation, verified by comparison of spectroscopic and chromatographic properties with an authentic standard synthesized by an unambiguous route.     

Xanthine, xanthosine and its nucleotides: solution structures of neutral and ionic forms, and relevance to substrate properties in various enzyme systems and metabolic pathways

The 6-oxopurine xanthine (Xan, neutral form 2,6-diketopurine) differs from the corresponding 6-oxopurines guanine (Gua) and hypoxanthine (Hyp) in that, at physiological pH, it consists of a approximately 1:1 equilibrium mixture of the neutral and monoanionic forms, the latter due to ionization of N(3)-H, in striking contrast to dissociation of the N(1)-H in both Gua and Hyp at higher pH. In xanthosine (Xao) and its nucleotides the xanthine ring is predominantly, or exclusively, a similar monoanion at physiological pH. The foregoing has, somewhat surprisingly, been widely overlooked in studies on the properties of these compounds in various enzyme systems and metabolic pathways, including, amongst others, xanthine oxidase, purine phosphoribosyltransferases, IMP dehydrogenases, purine nucleoside phosphorylases, nucleoside hydrolases, the enzymes involved in the biosynthesis of caffeine, the development of xanthine nucleotide-directed G proteins, the pharmacological properties of alkylxanthines. We here review the acid/base properties of xanthine, its nucleosides and nucleotides, their N-alkyl derivatives and other analogues, and their relevance to studies on the foregoing. Included also is a survey of the pH-dependent helical forms of polyxanthylic acid, poly(X), its ability to form helical complexes with a broad range of other synthetic homopolynucleotides, the base pairing properties of xanthine in synthetic oligonucleotides, and in damaged DNA, as well as enzymes involved in circumventing the existence of xanthine in natural DNA.     

Evaluation of the intramolecular stacking of the fluorosulfonylbenzoyl derivatives of 1,N6-ethenoadenosine, adenosine, and guanosine

The differences in conformation in solution of fluorosulfonylbenzoyl nucleosides were analyzed by fluorescence and proton nuclear magnetic resonance spectroscopy. The quantum yield of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine (5'-FSB epsilon A) in aqueous solution is low (? = 0.01) as compared to that of its parent nucleoside, ethenoadenosine (? = 0.54), and increases approximately 5-fold when measured in a series of solvents of decreasing dielectric constant. The quantum yield of 5'-p-sulfonylbenzoyl-1,N6-ethenoadenosine covalently bound to glutamate dehydrogenase and pyruvate kinase is also 0.01, suggesting that the analogue may exist in the same conformation when enzyme-bound as when free in solution. In D2O, the resonances of the purine ring protons on 5'-FSB epsilon A, 5'-p-fluorosulfonylbenzoyl adenosine (5'-FSBA), and 5'-p-fluorosulfonylbenzoyl guanosine (5'-FSBG) are shifted upfield by about 0.1-0.3 ppm relative to the corresponding protons of their parent nucleosides. The calculated difference in chemical shift (delta delta) decreases as the dielectric constant of the solvent decreases. The delta delta decreases with increasing temperature. These data indicate that 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG exist in aqueous solution in a conformation in which the purine ring is intramolecularly stacked with the benzoyl moiety. From the magnitude of change in delta delta for 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG as a function of solvent, it appears that the three analogues differ in their sensitivity to disruption of stacking. The solution conformation of these three fluorosulfonylbenzoyl nucleoside analogues may be an important determinant of their reaction with various enzymes and may explain differences among the analogues in their reaction with a single enzyme.     

Characterization of nucleoside transport systems in cultured rat epididymal epithelium

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na(+) dependent, while 30% was Na(+) independent. The Na(+)-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na(+)-dependent [(3)H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na(+)-dependent [(3)H]guanosine uptake was inhibited by thymidine by 20% and Na(+)-dependent [(3)H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na(+)-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.     

Unique substrate specificity of purine nucleoside phosphorylases from Thermus thermophilus

The degradation of purine nucleoside is the first step of purine nucleoside uptake. This degradation is catalyzed by purine nucleoside phosphorylase, which is categorized into two classes: hexameric purine nucleoside phosphorylase (6PNP) and trimeric purine nucleoside phosphorylase (3PNP). Generally, 6PNP and 3PNP degrade adenosine and guanosine, respectively. However, the substrate specificity of 6PNP and 3PNP of Thermus thermophilus (tt6PNP and tt3PNP, respectively) is the reverse of that anticipated based on comparison to other phosphorylases. Specifically, in this paper we reveal by gene disruption that tt6PNP and tt3PNP are discrete enzymes responsible for the degradation of guanosine and adenosine, respectively, in T. thermophilus HB8 cells. Sequence comparison combined with structural information suggested that Asn204 in tt6PNP and Ala196/Asp238 in tt3PNP are key residues for defining their substrate specificity. Replacement of Asn204 in tt6PNP with Asp changed the substrate specificity of tt6PNP to that of a general 6PNP. Similarly, substitution of Ala196 by Glu and Asp238 by Asn changed the substrate specificity of tt3PNP to that of a general 3PNP. Our results indicate that the residues at these positions determine substrate specificity of PNPs in general. Sequence analysis further suggested most 6PNP and 3PNP enzymes in thermophilic species belonging to the Deinococcus-Thermus phylum share the same critical residues as tt6PNP and tt3PNP, respectively.     

Synthesis of nucleoside libraries on solid support. II. 2,6,8-Trisubstituted purine nucleosides using 8-bromoguanosine as precursor

A series of 2,6,8-trisubstituted purine nucleoside libraries was prepared by parallel solid-phase synthesis using 8-bromoguanosine as a common synthetic precursor. Polystyrene-methoxytrityl chloride resin was linked to the N2 or O5' position of the guanosine analogues. 8-Bromoguanosine was derivatized at the C8 position via carbon-carbon bond formation. Nucleophilic aromatic substitution at C2 and/or C6 positions with various amines produced two series of purine nucleoside libraries with very diverse substitution.     

Identification and characterisation of high affinity nucleoside and nucleobase transporters in Toxoplasma gondii

The protozoan parasite Toxoplasma gondii depends upon salvaging the purines that it requires. We have re-analysed purine transport in T. gondii and identified novel nucleoside and nucleobase transporters. The latter transports hypoxanthine (TgNBT1; K(m)=0.91+/-0.19 microM) and is inhibited by guanine and xanthine: it is the first high affinity nucleobase transporter to be identified in an apicomplexan parasite. The previously reported nucleoside transporter, TgAT1, is low affinity with K(m) values of 105 and 134 microM for adenosine and inosine, respectively. We have now identified a second nucleoside transporter, TgAT2, which is high affinity and inhibited by adenosine, inosine, guanosine, uridine and thymidine (K(m) values 0.28-1.5 microM) as well as cytidine (K(i)=32 microM). TgAT2 also recognises several nucleoside analogues with therapeutic potential. We have investigated the basis for the broad specificity of TgAT2 and found that hydrogen bonds are formed with the 3' and 5' hydroxyl groups and that the base groups are bound through H-bonds with either N3 of the purine ring or N(3)H of the pyrimidine ring, and most probably pi-pi-stacking as well. The identification of these high affinity purine nucleobase and nucleoside transporters reconciles for the first time the low abundance of free nucleosides and nucleobases in the intracellular environment with the efficient purine salvage carried out by T. gondii.     

Synthesis, X-ray crystal structural study, antiviral and cytostatic evaluations of the novel unsaturated acyclic and epoxide nucleoside analogues

A series of the novel purine and pyrimidine nucleoside analogues were synthesised in which the sugar moiety was replaced by the 4-amino-2-butenyl (2-6 and 10-18) and oxiranyl (8 and 20) spacer. The Z- (2-6) and E-isomers (10-18) of unsaturated acyclic nucleoside analogues were synthesized by condensation of 2- and 6-substituted purine and 5-substituted uracil bases with Z- (1) or E-phthalimide (9) precursors. The oxiranyl nucleoside analogues (8 and 20) were obtained by epoxidation of 1 and 9 with m-chloroperoxybenzoic acid and subsequent coupling with adenine. The new compounds were evaluated for their antiviral and antitumor cell activities. Among the olefinic nucleoside analogues, Z-isomer of adenine containing 4-amino-2-butenyl side chain (6) exhibited the best cytostatic activities, particularly against colon carcinoma (SW 620, IC50 = 26 microM). Its E-isomer 15 did not show any antiproliferative activity against malignant tumor cell lines, except for a slight inhibition of colon carcinoma (SW 620, IC50 = 56.5 microM) cells. In general, Z-isomers showed better cytostatic activities than the corresponding E-isomers. (Z)-4-Amino-2-butenyl-adenine nucleoside analogue 6 showed albeit modest but selective activity against HIV-1 (EC50 = 4.83 microg mL(-1)).     

Transport of nucleosides in Bacillus subtilis: the effect of purine nucleosides on the cytidine-uptake

The structural effects of chemical modifications upon the affinity of purine nucleosides to cytidine-transport system in Bacillus subtilis were investigated using a series of modified derivatives. The interaction involves protein molecule(s) which require the presence and proper orientation of the sugar residue and its hydroxylic functions. Moreover, a specific interaction with the heterocyclic ring system is involved in the process which results in a requirement for an aromatic π -electron system and an absence of a polarizable function at position 6 of the purine heterocycle. The region in the protein responsible for the latter interaction is rather limited and, consequently, a proper nucleoside conformation is required.     

Modification of bovine pancreatic ribonuclease A with 6-chloropurine riboside

The chemical modification of bovine pancreatic ribonuclease A by 6-chloropurine riboside was studied to obtain information about the role of the purine nucleoside moiety of the ribonucleic acid in the enzyme-substrate interaction. The residues involved in the reaction were identified, after performic acid oxidation and trypsin digestion, by reverse-phase HPLC peptide mapping. The labeled peptides were detected by following the absorbance at 254 nm, and amino acid analyses of these peptides showed that the reaction had taken place with the amino groups of Lys-1, -37, -41, and -91. The specificity of the reaction was unaffected by changing the ligand:protein molar ratio. Partial separation of the reaction products was accomplished by means of chromatography on CM-Sepharose: four labeled fractions corresponding to mono- and bisubstituted derivatives were found. One of the monosubstituted fractions (fraction E) contained a homogeneous protein with the nucleoside bound to the alpha-amino group of Lys-1 whereas the other (fraction D) was a mixture of derivatives labeled in the epsilon-amino group of Lys-1, -37, -41, and -91. Kinetic studies of these two monosubstituted fractions were performed with cytidine 2',3'-phosphate and ribonucleic acid as substrates. These derivatives showed a noncompetitive inhibition-like behavior with respect to RNase A. Results support the existence of several RNase A regions with affinity for purine nucleosides.     

Acetylation and cleavage of purine nucleosides. Synthesis of 6-azauridine, 5-fluorouridine, and 5-methyluridine.

Inosine (I) when acetylated with acetic anhydride in the presence of acetyl chloride in acetic acid solution (the so called "acid acetylation"), affords an acetylated nucleoside III (75%) along with cleavage products of the nucleoside (hypoxanthine, 19%). The reaction of I with acetyl chloride (7 days) results in the formation of hypoxanthine (95%) and triacetylribofuranosyl chloride (IV) isolated in the form of tetraacetylribofuranose (47%). The acetylated purine nucleoside affords a similar result by reaction with acetyl chloride or acetyl bromide. 2'-Deoxyuridine gives a diacetyl derivative (80%) by reaction with acetyl bromide. On treatment with acetyl bromide, the nucleoside bond of purine nucleosides is quantitatively cleavaged (4 h, 20 degrees C) with the formation of tri-O-acetyl-D-ribofuranosyl bromide (X). The halogenose X affords pure beta-anomers, namely, 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose (75%), the triacetyl derivatives of 5-methyluridine (XVIIa; 75%, referred to guanosine), 6-azauridine (XVIII; 71%), and 5-fluorouridine (XIXa; 75%).     

Catabolic pathways of purine ribonucleotides and deoxyribonucleotides in lymphocytes

Deficiency of either one of the subsequent purine catabolic enzymes adenosine deaminase or purine nucleoside phosphorylase results in immunodeficiency disease in humans. However, the mechanism by which impairment of purine metabolism may cause immunodeficiency is unclear. In the present work we have studied the catabolism of purine ribonucleotides and deoxyribonucleotides in T lymphocytes to better understand the role of purine nucleoside phosphorylase and adenosine deaminase in the immune function. It was found that purine deoxyribonucleotides are degraded via catabolic pathways distinctly different from those used for purine ribonucleotide degradation. Thus both adenine and guanine ribonucleotides are deaminated to IMP whereas purine deoxyribonucleotides are exclusively dephosphorylated to the corresponding deoxyribonucleosides. These findings may explain the relatively higher degradation rates of purine deoxyribonucleotides in mammalian cells as compared to purine ribonucleotides. The catabolism of purine nucleotides is tightly linked to the active purine nucleoside cycles which consist of the phosphorolysis of purine nucleosides and deoxyribonucleosides to their corresponding bases, their salvage to monophosphates and back to the corresponding ribonucleosides. The above observations also imply that a possible role of the purine nucleoside cycles is to convert purine deoxyribonucleotides into their corresponding ribonucleotide derivatives. Deficiencies of purine nucleoside phosphorylase or of adenosine deaminase activities, enzymes which participate or lead to the purine nucleoside cycles, thus result in a selective impaired deoxyribonucleotide catabolism and immunodeficiency.