scholarly journals Efficient Synthesis of Purine Nucleoside Analogs by a New Trimeric Purine Nucleoside Phosphorylase from Aneurinibacillus migulanus AM007

Molecules ◽  
2019 ◽  
Vol 25 (1) ◽  
pp. 100
Author(s):  
Gaofei Liu ◽  
Tiantong Cheng ◽  
Jianlin Chu ◽  
Sui Li ◽  
Bingfang He
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Nucleoside Analogs ◽  
Industrial Applications ◽  
Purine Nucleoside ◽  
Initial Activity ◽  
One Pot ◽  
Purine Base ◽  
Nucleoside Phosphorylase ◽  
Pyrimidine Nucleoside Phosphorylase ◽  
Nucleoside Phosphorylases

Purine nucleoside phosphorylases (PNPs) are promising biocatalysts for the synthesis of purine nucleoside analogs. Although a number of PNPs have been reported, the development of highly efficient enzymes for industrial applications is still in high demand. Herein, a new trimeric purine nucleoside phosphorylase (AmPNP) from Aneurinibacillus migulanus AM007 was cloned and heterologously expressed in Escherichia coli BL21(DE3). The AmPNP showed good thermostability and a broad range of pH stability. The enzyme was thermostable below 55 °C for 12 h (retaining nearly 100% of its initial activity), and retained nearly 100% of the initial activity in alkaline buffer systems (pH 7.0–9.0) at 60 °C for 2 h. Then, a one-pot, two-enzyme mode of transglycosylation reaction was successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP) derived from Brevibacillus borstelensis LK01 and AmPNP for the production of purine nucleoside analogs. Conversions of 2,6-diaminopurine ribonucleoside (1), 2-amino-6-chloropurine ribonucleoside (2), and 6-thioguanine ribonucleoside (3) synthesized still reached >90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio of BbPyNP: AmPNP (2:20 μg/mL). Thus, the new trimeric AmPNP is a promising biocatalyst for industrial production of purine nucleoside analogs.

FISH-MUSCLE PURINE AND PYRIMIDINE NUCLEOSIDE PHOSPHORYLASES

1967 ◽  
Vol 45 (3) ◽  
pp. 409-419 ◽  
Author(s):  
H. L. A. Tarr ◽  
Joan E. Roy
Keyword(s):  
Enzyme Preparation ◽  
Purine Nucleoside Phosphorylase ◽  
Deae Cellulose ◽  
Fish Muscle ◽  
Purine Nucleoside ◽  
Aqueous Extracts ◽  
Pyrimidine Nucleoside ◽  
Nucleoside Phosphorylase ◽  
Pyrimidine Nucleoside Phosphorylase ◽  
Nucleoside Phosphorylases

Three purine nucleoside phosphorylase preparations (isoenzymes) were obtained by ammonium sulfate fractionation and DEAE-cellulose chromatography of aqueous extracts of lingcod muscle. Dialysis, adsorption on alumina Cγ, and elution with 0.4 M phosphate buffer yielded further purification. The most active enzyme preparation had about 120 times the activity of initial extracts. It utilized hypoxanthine, 6-mercaptopurine, guanine, 8-azaguanine, xanthine, adenine, 2,6-diaminopurine and 6-methylpurine in presence of ribose 1-phosphate or deoxyribose 1-phosphate. Several substituted purines were not utilized and did not inhibit the reaction between hypoxanthine and the pentose phosphates. The Kmwith inosine as substrate was 3.2 × 10−6 M. A pyrimidine nucleoside phosphorylase, distinct from the purine nucleoside phosphorylase, occurred in the DEAE-cellulose fraction comprising one of the purine nucleoside phosphorylases. Its activity was much lower than that of the purine nucleoside phosphorylase preparations. Uridine and thymidine were the best substrates. Deoxyuridine was a poor substrate, and neither cytidine nor deoxycytidine was utilized. The equilibrium with all preparations was about 80% in favor of nucleoside formation. The purified enzymes were all destroyed by freezing.

Localization of purine and pyrimidine nucleoside phosphorylases in heart, kidney, and liver

1980 ◽  
Vol 239 (6) ◽  
pp. H721-H730 ◽  
Author(s):  
R. Rubio ◽  
R. M. Berne
Keyword(s):  
Guinea Pig ◽  
Kupffer Cells ◽  
Cellular Localization ◽  
Purine Nucleoside Phosphorylase ◽  
Degradation Products ◽  
Purine Nucleoside ◽  
Capillary Endothelium ◽  
Cell Fractionation ◽  
Nucleoside Phosphorylase ◽  
Nucleoside Phosphorylases

In isolated livers and kidneys perfused with Krebs-Henseleit solution, the relationship of the concentration of adenosine (Ado) to that of its degradation products inosine (Ino) and hypoxanthine (Hyp) in biliary, urinary, and venous effluents were determined. They revealed ratios of Hyp:Ado:Ino, 1.9:1:0.9, 0.7:1:0.6, and 1.3:1:0.5 for guinea pig biliary, guinea pig urinary, and rat urinary effluents, respectively, and their respective venous effluent were 58:1:29, 8.6:1:5.4, and 7.4:1:3.2. The greater proportion of Ino and Hyp in the venous effluents suggests active production in Ino and Hyp at the vessel wall. Purine nucleoside phosphorylase localization was determined histochemically and found most active in the cytoplasm of capillary endothelium and Kupffer cells. Thus, there is agreement between purine analysis and histochemical findings. The reliability of the histochemical technique was also tested by comparing activities of purine nucleoside phosphorylase (a cytoplasmic enzyme) and pyrmidine nucleoside phosphorylase (a nuclear enzyme) that catalyze similar reactions (nucleoside + inorganic phosphate in equilibrium base + ribose-1-phosphate) but with different base specificites and cellular localization, as indicated by cell fractionation studies. The histochemical results show that in contrast to the purine nucleoside phosphorylase, the pyrmidine specific enzyme was most active in the nuclei of endothelial and Kupffer cells. Thus, the technique discriminates between the two enzymes.

scholarly journals Identification of a Subversive Substrate of Trichomonas vaginalis Purine Nucleoside Phosphorylase and the Crystal Structure of the Enzyme-Substrate Complex

2005 ◽  
Vol 280 (23) ◽  
pp. 22318-22325 ◽  
Author(s):  
Yang Zang ◽  
Wen-Hu Wang ◽  
Shaw-Wen Wu ◽  
Steven E. Ealick ◽  
Ching C. Wang
Keyword(s):  
Active Site ◽  
Trichomonas Vaginalis ◽  
Purine Nucleoside Phosphorylase ◽  
Quaternary Structure ◽  
Transmitted Disease ◽  
Purine Nucleoside ◽  
Purine Base ◽  
Nucleoside Phosphorylase ◽  
E Coli

Trichomonas vaginalis is an anaerobic protozoan parasite that causes trichomoniasis, a common sexually transmitted disease with worldwide impact. One of the pivotal enzymes in its purine salvage pathway, purine nucleoside phosphorylase (PNP), shows physical properties and substrate specificities similar to those of the high molecular mass bacterial PNPs but differing from those of human PNP. While carrying out studies to identify inhibitors of T. vaginalis PNP (TvPNP), we discovered that the nontoxic nucleoside analogue 2-fluoro-2′-deoxyadenosine (F-dAdo) is a “subversive substrate.” Phosphorolysis by TvPNP of F-dAdo, which is not a substrate for human PNP, releases highly cytotoxic 2-fluoroadenine (F-Ade). In vitro studies showed that both F-dAdo and F-Ade exert strong inhibition of T. vaginalis growth with estimated IC50 values of 106 and 84 nm, respectively, suggesting that F-dAdo might be useful as a potential chemotherapeutic agent against T. vaginalis. To understand the basis of TvPNP specificity, the structures of TvPNP complexed with F-dAdo, 2-fluoroadenosine, formycin A, adenosine, inosine, or 2′-deoxyinosine were determined by x-ray crystallography with resolutions ranging from 2.4 to 2.9 Å. These studies showed that the quaternary structure, monomer fold, and active site are similar to those of Escherichia coli PNP. The principal active site difference is at Thr-156, which is alanine in E. coli PNP. In the complex of TvPNP with F-dAdo, Thr-156 causes the purine base to tilt and shift by 0.5 Å as compared with the binding scheme of F-dAdo in E. coli PNP. The structures of the TvPNP complexes suggest opportunities for further improved subversive substrates beyond F-dAdo.

DESIGN AND EVALUATION OF 5′-MODIFIED NUCLEOSIDE ANALOGS AS PRODRUGS FOR AN E. COLI PURINE NUCLEOSIDE PHOSPHORYLASE MUTANT

2005 ◽  
Vol 24 (5-7) ◽  
pp. 387-392 ◽  
Author(s):  
William B. Parker ◽  
Paula W. Allan ◽  
Steve E. Ealick ◽  
Eric J. Sorscher ◽  
Abdalla E. A. Hassan ◽  
...  
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Nucleoside Analogs ◽  
Purine Nucleoside ◽  
Nucleoside Phosphorylase ◽  
E Coli ◽  
Modified Nucleoside

Purine Nucleoside Phosphorylase. 3. Reversal of Purine Base Specificity by Site-Directed Mutagenesis†

Biochemistry ◽  
1997 ◽  
Vol 36 (39) ◽  
pp. 11749-11756 ◽  
Author(s):  
Johanna D. Stoeckler ◽  
Anne F. Poirot ◽  
Rose Marie Smith ◽  
Robert E. Parks, ◽  
Steven E. Ealick ◽  
...  
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Site Directed Mutagenesis ◽  
Purine Nucleoside ◽  
Directed Mutagenesis ◽  
Purine Base ◽  
Nucleoside Phosphorylase ◽  
Base Specificity

The Chemoenzymatic Synthesis of 2-Chloro- and 2-Fluorocordycepins

Synthesis ◽  
2017 ◽  
Vol 49 (21) ◽  
pp. 4853-4860 ◽  
Author(s):  
Igor Mikhailopulo ◽  
Alexandra Denisova ◽  
Yulia Tokunova ◽  
Ilja Fateev ◽  
Alexandra Breslav ◽  
...  
Keyword(s):  
Efficient Method ◽  
Purine Nucleoside Phosphorylase ◽  
Chemoenzymatic Synthesis ◽  
Purine Nucleoside ◽  
Limiting Factor ◽  
One Pot ◽  
Nucleoside Phosphorylase ◽  
E Coli ◽  
Poor Solubility ◽  
Practical Synthesis

Two approaches to the chemoenzymatic synthesis of 2-fluorocordycepin and 2-chlorocordycepin were studied: (i) the use of 3′-deoxyadenosine (cordycepin) and 3′-deoxyinosine (3′dIno) as donors of 3-deoxy-d-ribofuranose in the transglycosylation of 2-fluoro- (2FAde) and 2-chloroadenine (2ClAde) catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP), and (ii) the use of 2-fluoroadenosine and 3′-deoxyinosine as substrates of the cross-glycosylation and PNP as a biocatalyst. An efficient method for 3′-deoxyinosine synthesis starting from inosine was developed. However, the very poor solubility of 2ClAde and 2FAde is the limiting factor of the first approach. The second approach enables this problem to be overcome and it appears to be advantageous over the former approach from the viewpoint of practical synthesis of the title nucleosides. The 3-deoxy-α-d-ribofuranose-1-phosphate intermediary formed in the 3′dIno phosphorolysis by PNP was found to be the weak and marginal substrate of E. coli thymidine (TP) and uridine (UP) phosphorylases, respectively. Finally, one-pot cascade transformation of 3-deoxy-d-ribose in cordycepin in the presence of adenine and E. coli ribokinase, phosphopentomutase, and PNP was tested and cordycepin formation in ca. 3.4% yield was proved.

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