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

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.

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.

Synthesis of 6-Aryloxy- and 6-Arylalkoxy-2-chloropurines and Their Interactions with Purine Nucleoside Phosphorylase from Escherichia coli

1999 ◽  
Vol 54 (12) ◽  
pp. 1055-1067 ◽  
Author(s):  
Agnieszka Bzowska ◽  
Lucyna Magnowska ◽  
Zygmunt Kazimierczuk
Keyword(s):  
Mixed Type ◽  
Purine Nucleoside Phosphorylase ◽  
Phase Transfer ◽  
Competitive Inhibition ◽  
Purine Nucleoside ◽  
Inhibition Constant ◽  
Purine Base ◽  
Nucleoside Phosphorylase ◽  
E Coli ◽  
Inhibition Constants

The phase transfer method was applied to perform the nucleophilic substitution of 2,6- dichloropurines by modified arylalkyl alcohol or phenols. Since under these conditions only the 6-halogen is exchanged, this method gives 2-chloro-6-aryloxy- and 2-chloro-6-arylalkoxypurines. 2-Chloro-6-benzylthiopurine was synthesized by alkylation of 2-chloro-6-thiopurine with benzyl bromide. The stereoisomers of 2-chloro-6-(1-phenyl-1-ethoxy)purine were obtained from R- and S-enantiomers of sec.-phenylethylalcohol and 2,6-dichloropurine. All derivatives were tested for inhibition with purified hexameric E. coli purine nucleoside phosphorylase (PNP). For analogues showing IC50 < 10 μm, the type of inhibition and inhibition constants were determined. In all cases the experimental data were best described by the mixed-type inhibition model and the uncompetitive inhibition constant, Kiu, was found to be several-fold lower than the competitive inhibition constant, Kic. This effect seems to be due to the 6-aryloxy- or 6-arylalkoxy substituent, because a natural PNP substrate adenine, as well as 2-chloroadenine, show mixed type inhibition with almost the same inhibition constants Kiu and KiC. The most potent inhibition was observed for 6-benzylthio-2-chloro-, 6-benzyloxy-2-chloro-, 2-chloro-6-(2-phenyl-l-ethoxy), 2-chloro-6-(3-phenyl-l-propoxy)- and 2-chloro-6-ethoxypurines (Kiu = 0.4, 0.6, 1.4, 1.4 and 2.2 μm, respectively). The R-stereoisomer of 2-chloro-6-(1pheny-1-ethoxy)purine has Kiu = 2.0 μm, whereas inhibition of its S counterpart is rather weak (IC50> 12 μm). More rigid (e.g. phenoxy-), non-planar (cyclohexyloxy-), or more bulky (2,4,6-trimethylphenoxy-) substituents at position 6 of the purine base gave less potent inhibitors (IC50 = 26, 56 and >100 μm, respectively). The derivatives are selective inhibitors of hexameric “high-molecular mass” PNPs because no inhibitory activity vs. trimeric Cellulomonas sp. PNP was detected. By establishing the ligand-dependent stabilization pattern of the E. coli PNP it was shown that the new derivatives, similarly as the natural purine bases, are able to form a dead-end ternary complex with the enzyme and orthophosphate. It was also shown that the derivatives are substrates in the reverse synthetic direction catalyzed by E. coli PNP

Purine nucleoside phosphorylase: Isolation and characterization

1990 ◽  
Vol 55 (12) ◽  
pp. 2987-2999 ◽  
Author(s):  
Katarina Šedivá ◽  
Ivan Votruba ◽  
Antonín Holý ◽  
Ivan Rosenberg
Keyword(s):  
Molecular Weight ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Leukemia Cells ◽  
Ph Optimum ◽  
Nucleoside Phosphorylase ◽  
Isolation And Characterization ◽  
Reaction Proceeds ◽  
The Matrix ◽  
Sepharose 4B

Purine nucleoside phosphorylase (PNP) from mouse leukemia cells L1210 was purified to homogeneity by a combination of ion exchange and affinity chromatography using AE-Sepharose 4B and 9-(p-succinylaminobenzyl)hypoxanthine as the matrix and the ligand, respectively. The native enzyme has a molecular weight of 104 000 and consists of three subunits of equal molecular weight of 34 000. The results of isoelectric focusing showed that the enzyme is considerably microheterogeneous over the pI-range 4.0-5.8 and most likely consists of eight isozymes. The temperature and pH-optimum of phosphorolysis, purine nucleoside synthesis and also of transribosylation is identical, namely 55 °C and pH 7.4. The transribosylation reaction proceeds in the presence of phosphate only. The following Km-values (μmol l-1) were determined for phosphorolysis: inosine 40, 2'-deoxyinosine 47, guanosine 27, 2'-deoxyguanosine 32. The Km-values (μmol l-1) of purine riboside and deoxyriboside synthesis are lower than the values for phosphorolysis (hypoxanthine 18 and 34, resp., guanine 8 and 11, resp.). An affinity lower by one order shows PNP for (-D-ribose-1-phosphate, (-D-2-deoxyribose-1-phosphate (Km = 200 μmol l-1 in both cases) and phosphate (Km = 805 μmol l-1). The substrate specificity of the enzyme was also studied: positions N(1), C(2) and C(8) are decisive for the binding of the substrate (purine nucleoside).

Purine Nucleoside Phosphorylase (PNP) Deficiency Leading to Accumulation of Lymphocytes in S-Phase

1986 ◽  
Vol 3 (4) ◽  
pp. 353-359 ◽  
Author(s):  
Ger T. Rijkers ◽  
Ben J. M. Zegers ◽  
Leo J. M. Spaapen ◽  
Derk H. Rutgers ◽  
John J. Roord ◽  
...  
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
S Phase ◽  
Purine Nucleoside ◽  
Nucleoside Phosphorylase
Sign in / Sign up

Export Citation Format

Share Document