Low-molecular-mass purine nucleoside phosphorylase: characterization and application in enzymatic synthesis of nucleoside antiviral drugs

Purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of various purine nucleosides is widely distributed in prokaryotes and eukaryotes. Four pnp genes from Bacillus subtilis 168, Escherichia coli K-12 and Pseudoalteromonas sp. XM2107 were cloned by PCR and expressed in E. coli XL1-Blue. Recombinant PNPs (rPNPs) were purified by Ni²⁺-NTA chromatography. Compared with other rPNPs, PNP₈₁₆ was a low-molecular-mass homotrimer, which exhibited 11-, 4- and 1.5-fold higher values in k _cat/K _m using inosine as the substrate at 37°C. The PNP₈₁₆ or engineered strain XBlue (pQE-816) had a higher catalytic activity than other rPNPs or engineered strains during the enzymatic synthesis of ribavirin, which suggested that the low-molecular-mass homotrimer derived from microorganisms has higher catalytic activity for synthesis of nucleoside antiviral drugs.     

A second purine nucleoside phosphorylase in Escherichia coli K-12

Summary The presence of a second purine nucleoside phosphorylase in wild-type strains of E. coli K-12 after growth on xanthosine has been demonstrated. Like other purine nucleoside phosphorylases it is able to carry out both phosphorylosis and synthesis of purine deoxy- and ribonucleosides whilst pyrimidine nucleosides cannot act as substrates. In contrast to the well characterised purine nucleoside phosphorylase of E. coli K-12 (encoded by the deoD gene) this new enzyme could act on xanthosine and is hence called xanthosine phosphorylase. Studies of its substrate specificity showed that xanthosine phosphorylase, like the mammalian purine nucleoside phosphorylases, has no activity towards adenine and the corresponding nucleosides. Determinations of K _m and gel filtration behaviour was carried out on crude dialysed extracts. The presence of xanthosine phosphorylase enables E. coli to grow on xanthosine as carbon source. Xanthosine was the only compound found which induced xanthosine phosphorylase. No other known nucleoside catabolising enzyme was induced by xanthosine. The implications of non-linear induction kinetics of xanthosine phosphorylase is discussed.     

Molecular modeling and dynamics studies of purine nucleoside phosphorylase from Bacteroides fragilis

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides, except adenosine, to generate ribose 1-phosphate and the purine base. This work describes for the first time a structural model of PNP from Bacteroides fragilis (Bf). We modeled the complexes of BfPNP with six different ligands in order to determine the structural basis for specificity of these ligands against BfPNP. Comparative analysis of the model of BfPNP and the structure of HsPNP allowed identification of structural features responsible for differences in the computationally determined ligand affinities. The molecular dynamics (MD) simulation was assessed to evaluate the overall stability of the BfPNP model. The superposition of the final onto the initial minimized structure shows that there are no major conformational changes from the initial model, which is consistent with the relatively low root mean square deviation (RMSD). The results indicate that the structure of the model was stable during MD, and does not exhibit loosely structured loop regions or domain terminals.     

Characterization of a recombinant cold-adapted purine nucleoside phosphorylase and its application in ribavirin bioconversion

Ribavirin is a broad-spectrum antiviral drug and can be produced by enzymatic synthesis by purine nucleoside phosphorylase (PNP). In this study, we describe the application of such a cold-adapted XmPNP in ribavirin bioconversion which showed approximately 15°C lower optimum temperature and 1.80-fold higher catalytic efficiency (k_cat/K_m) at 37°C within substrate inosine than homolog in E. coli. By contrast, E. coli (XmPNP) took only 12 h to reach maximum substrate conversion rate (70%) under its optimum temperature (50°C) by using recombinant strain cell as enzyme source, but E. coli (EcPNP) did at 24 h. These results suggest cold-adapted PNP is one attractive candidate for ribavirin bioconversion and other nucleoside medications to improve the catalytic efficiency.     

Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus

Adenosine phosphorylase, a purine nucleoside phosphorylase endowed with high specificity for adenine nucleosides, was purified 117-fold from vegetative forms of Bacillus cereus. The purification procedure included ammonium sulphate fractionation, pH 4 treatment, ion exchange chromatography on DEAE-Sephacel, gel filtration on Sephacryl S-300 HR and affinity chromatography on N⁶-adenosyl agarose. The enzyme shows a good stability to both temperature and pH. It appears to be a homohexamer of 164 ± 5 kDa. Kinetic characterization confirmed the specificity of this phosphorylase for 6-aminopurine nucleosides. Adenosine was the preferred substrate for nucleoside phosphorolysis (k_cat/K_m 2.1 × 10⁶ s^− 1 M^− 1), followed by 2′-deoxyadenosine (k_cat/K_m 4.2 × 10⁵ s^− 1 M^− 1). Apparently, the low specificity of adenosine phosphorylase towards 6-oxopurine nucleosides is due to a slow catalytic rate rather than to poor substrate binding.     

Guanosine triphosphate catabolism in purine nucleoside phosphorylase deficient human B lymphoblastoid cells

GTP catabolism induced by sodium azide or deoxyglucose was studied in purine nucleoside phosphorylase (PNP) deficient human B lymphoblastoid cells. In PNP deficient cells, as in control cells, guanylate was both dephosphorylated and deaminated but dephosphorylation was the major pathway. Only nucleosides were excreted during GTP catabolism by PNP deficient cells and the main product was guanosine. The level of nucleoside excretion was largely affected by intracellular orthophosphate (P_i) level. In contrast, normal cells excreted nucleosides only at low P_i level while at high P_i levels, purine bases (guanine and hypoxanthine) were exclusively excreted. PNP deficiency had no effect on the extent of GMP deamination.     

Identification and characterization of two adenosine phosphorylase activities in Mycobacterium smegmatis

Purine nucleoside phosphorylase (PNP) is an important enzyme in purine metabolism and cleaves purine nucleosides to their respective bases. Mycobacterial PNP is specific for 6-oxopurines and cannot account for the adenosine (Ado) cleavage activity that has been detected in M. tuberculosis and M. smegmatis cultures. In the current work, two Ado cleavage activities were identified from M. smegmatis cell extracts. The first activity was biochemically determined to be a phosphorylase that could reversibly catalyze adenosine + phosphate ↔ adenine + alpha-d-ribose-1-phosphate. Our purification scheme led to a 30-fold purification of this activity, with the removal of more than 99.9% of total protein. While Ado was the preferred substrate, inosine and guanosine were also cleaved, with 43% and 32% of the Ado activity, respectively. Our data suggest that M. smegmatis expresses two PNPs: a previously described trimeric PNP that can cleave inosine and guanosine only and a second, novel PNP (Ado-PNP) that can cleave Ado, inosine, and guanosine. Ado-PNP had an apparent K_m (K_{m app}) of 98 ± 6 μM (with Ado) and a native molecular mass of 125 ± 7 kDa. The second Ado cleavage activity was identified as 5′-methylthioadenosine phosphorylase (MTAP) based on its biochemical properties and mass spectrometry analysis. Our study marks the first report of the existence of MTAP in any bacterium. Since human cells do not readily convert Ado to Ade, an understanding of the substrate preferences of these enzymes could lead to the identification of Ado analogs that could be selectively activated to toxic products in mycobacteria.     

A New Synthesis of Hepialone [(R)-2-Ethyl-6-methyl-2,3-dihydro-4H-pyran-4-one], the Principal Component from Male Sex Scales of the Ghost Moth,Hepialus californicus B

Microorganisms that produce 5-methyluridine (ribothymidine) directly from purine nucleosides and thymine were screened from our stock cultures. Of the 400 strains tested, Erwinia carotovora AJ- 2992 was found to possess the most potent ability as to production of 5-methyluridine from guanosine and thymine. In the presence of intact cells of Er. carotovora AJ-2992 as the enzyme source, 222 mm 5-methyluridine was produced from 300 mm guanosine and 300 mm thymine at 60°C on 48 hr incubation. The enzymatic production of 5-methyluridine by Er. carotovora AJ-2992 was found to involve the following two successive reactions via ribose-1-phosphate as an intermediate: phosphorolysis of purine nucleosides to ribose-1-phosphate and purine bases by purine nucleoside phosphorylase, followed by condensation of ribose-1-phosphate and thymine into 5-methyluridine by pyrimidine nucleoside phosphorylase.     

Interactions of Calf Spleen Purine Nucleoside Phosphorylase with Formycin B and its Aglycone—Spectroscopic and Kinetic Studies

Phosphorolysis of 7-methylguanosine by calf spleen purine nucleoside phosphorylase (PNP) is weakly inhibited, uncompetitively, by Formycin B (FB) with K _i = 100 μ M and more effectively by its aglycone (7KPP), IC₅₀ 35–100 μ M. In striking contrast, 7KPP inhibits the reverse reaction (synthesis of 8-azaguanosine from 8-azaguanine) competitively, with K _i ～ 2–4 μ M. Formycin B forms only a weakly fluorescent complex with PNP, and 7KPP even less so, indicating that both ligands bind as the neutral, not anionic, forms. 7KPP is a rare example of a PNP non-substrate inhibitor of both the phosphorolytic and reverse synthetic pathways.     

Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi

A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K_m values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K_i and K_i′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn²⁺ or Co²⁺ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH₂-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.     

The dipeptidyl carboxypeptidase of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue: overproduction and molecular characterization of the recombinant enzyme

To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His₆-tagged EcDCP (His₆-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity by Ni²⁺-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His₆-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His₆-EcDCP was stimulated by 1.5 fold. The K _M and k _cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s⁻¹, respectively. His₆-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His₆-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17% α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.     

Two fluorogenic substrates for purine nucleoside phosphorylase,selective for mammalian and bacterial forms of the enzyme

Two nontypical nucleosides, 7-β-d-ribosyl-2,6-diamino-8-azapurine and 8-β-d-ribosyl-2,6-diamino-8-azapurine, have been found to exhibit moderately good, and selective, substrate properties toward calf and bacterial (Escherichia coli) forms of purine nucleoside phosphorylase (PNP). The former compound is effectively phosphorolysed by calf PNP and the latter by PNP from E. coli. Both compounds are fluorescent with λ_max ∼ 425 to 430 nm, but the reaction product, 2,6-diamino-8-azapurine, emits in a different spectral region (λ_max ∼ 363 nm) with nearly 40% yield, providing a strong fluorogenic effect at 350 to 360 nm.     

X-Ray Crystallographic and Kinetic Analysis of Human Purine Nucleoside Phosphorylase Complexes with 1-β-D-Ribofuranosyl-1,2,4-triazole-3-carobxamide and 1-β-D-Ribofuranosyl-1,2,4-triazole-3-carboxamidine

Abstract

The three-dimensional structures of the complexes between human erythrocytic purine nucleoside phosphorylase (PNP) and both 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) and 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine (TCNR) have been determined using X-ray crystallographic techniques. The structures have been refined at 2.9 Å resolution using simulated annealing and conjugate-gradient minimization techniques to an R value of 21.8% for ribavirin and 20.8% for TCNR. Ribavirin and TCNR are truncated nucleosides corresponding to adenosine and inosine, respectively, and are of potential interest as PNP inhibitors. Kinetic parameters have been determined for recombinant wild-type PNP and for a mutant PNP in which Asn 243 is converted to Asp. The K_i value for ribavirin is 4.9 mM with wild-type PNP and 4.7 mM with the Asn243Asp mutant, while the K_i values for TCNR are 17.6 μM and 3.8 μM with wild-type and mutant, respectively. X-ray crystallographic studies showed that the binding geometry for both of these substrate analogues was similar to that seen for natural substrates. The glycosidic torsion angles (χ) were ?34° for ribavirin and ?39° for TCNR which are in good agreement with values seen for other studied nucleoside complexes with PNP, but which are unusual when compared to those seen for free nucleic acid derivatives. Based upon the three-dimensional structure, interactions of Asn 243 and Glu 201 with a protonated carboxamidine of TCNR explain the stronger inhibition of PNP observed for TCNR over ribavirin.     

Inosine nucleosidase from Azotobacter vinelandii

An enzyme catalyzing the hydrolysis of purine nucleosides was found to occur in the extract of Azotobacter vinelandii, strain 0, and was highly purified by ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxylapatite chromatography and gel filtration on Sephadex G-150. A strict substrate specificity of the purified enzyme was shown with respect to the base components. The enzyme specifically attacked the nucleosides without amino groups in the purine moiety: inosine gave the maximum rate of hydrolysis and xanthosine was hydrolyzed to a lesser extent. The pH optimum of inosine hydrolysis was observed from pH 7 to 9, while xanthosine was hydrolyzed maximally at pH 7. The K _m values of the enzyme for inosine were 0.65 and 0.85 mM at pH 7.1 and 9.0, respectively, and the value for xanthosine was 1.2 mM at pH 7.1.Several nucleotides inhibited the enzyme: the phosphate portions of the nucleotides were suggested to be responsible for the inhibition by nucleotides. Although the inhibition of the enzyme by nucleotides was apparently non-competitive type with respect to inosine, allosteric (cooperative) binding of the substrate was suggested in the presence of the inhibitor. The physiological significance of the enzyme was discussed in connection with the degradation and salvage pathways of purine nucleotides.     

Cloning,characterization and expression of the chitinase gene of <Emphasis Type="Italic">Enterobacter</Emphasis> sp. NRG4

A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg⁻¹. It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the K_m, k_cat and catalytic efficiency (k_cat/K_m) values of recombinant chitinase were found to be 1.27 mg ml⁻¹, 0.69 s⁻¹ and 0.54 s⁻¹M⁻¹ respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.     

Fluorescence studies of calf spleen purine nucleoside phosphorylase (PNP) complexes with guanine and 9-deazaguanine

Interactions of trimeric calf spleen purine nucleoside phosphorylase (PNP) with guanine (Gua) and its analogue, 9-deazaguanine (9-deaza-Gua), were studied by means of the steady-state fluorescence. The aim was to test the hypothesis that the enzyme stabilizes the anionic form of purine, inferred previously from the unusual increase of fluorescence observed after binding of guanine by calf spleen PNP. We have found that the dissociation constants obtained form titration experiments are in fact pH-independent in the range 7.0-10.25 for both PNP/Gua and PNP/9-deaza-Gua complexes. In particular, at pH 7.0 we found K _d = 0.12 ± 0.02 μ M for Gua and 0.16 ± 0.01 μ M for 9-deaza-Gua, while at the conditions where there is more than 40% of the anionic form the respective values were K _d = 0.15 ± 0.01 μ M for Gua (pH 9.0) and 0.25 ± 0.02 μ M for 9-deaza-Gua (pH 10.25). Hence, the enzyme does not prefer binding of anionic forms of these ligands in respect to the neutral ones. This result questions the involvement of the anionic forms in the reaction catalyzed by trimeric PNPs, and contradicts the hypothesis of a strong hydrogen bond formation between the enzyme Asn 243 residue and the purine N(7) position.     

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.     

Cloning and expression of the sucrose phosphorylase gene from <Emphasis Type="Italic">Leuconostoc </Emphasis><Emphasis Type="Italic">mesenteroides</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K _m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl moiety of sucrose to cytosine monophosphate (CMP). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phosphorylase-mediated mobilization of the amino group of adenine in Bacillus cereus

Mobilization of the ribose moiety of purine nucleosides as well as of the amino group of adenine may be realized in Bacillus cereus by the concerted action of three enzymes: adenosine phosphorylase, adenosine deaminase, and purine nucleoside phosphorylase. In this pathway, ribose-1-phosphate and inorganic phosphate act catalytically, being continuously regenerated by purine nucleoside phosphorylase and adenosine phosphorylase, respectively. As a result of such a metabolic pathway, adenine is quantitatively converted into hypoxanthine, thus overcoming the lack of adenase in B. cereus.     

Synthesis of 2-Chloro-6-aryloxy- and 2-Chloro-6-alkoxyarylpurines and Their Properties in the Purine Nucleoside Phosphorylase (PNP) System

Abstract

A series of 2-chloro-6-aryloxy- and 2-chloro-6-alkoxyarylpurines was synthesized and their kinetic properties in the purine nucleoside phosphorylase (PNP) system were determined. All compounds showed inhibitory activity (IC₅₀ in the range 0.5-76 μM) vs. hexameric (“high-molecular weight”) PNP from E. coli. By contrast, no inhibition vs. trimeric Cellulomonas PNP was detected.