Validation of the catalytic mechanism of Escherichia coli purine nucleoside phosphorylase by structural and kinetic studies

Mikleušević, Goran; Štefanić, Zoran; Narczyk, Marta; Wielgus‐Kutrowska, Beata; Bzowska, Agnieszka; Luić, Marija

doi:10.1016/j.biochi.2011.05.030

Cited by 38 publications

(87 citation statements)

References 23 publications

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“…This finding is in agreement with the observation that all active sites in the Arg24Ala mutant complexed with P i are open with loosely bound phosphate anion. These remarkable difference even made us suspicious about the binding of the P i by the Arg24Ala mutant, although it was previously shown by measuring effects of the ligand on the thermal stability of the enzyme [21]. Moreover, binding must take place since the mutant exhibits activity, albeit small (0.2%-0.6% that of the WT) versus natural substrates, and good activity versus 7-methylguanosine (30% that of the WT) [21].…”

Section: Phosphate Binding Is Affected By Arg24ala Mutation and Activmentioning

confidence: 99%

“…These remarkable difference even made us suspicious about the binding of the P i by the Arg24Ala mutant, although it was previously shown by measuring effects of the ligand on the thermal stability of the enzyme [21]. Moreover, binding must take place since the mutant exhibits activity, albeit small (0.2%-0.6% that of the WT) versus natural substrates, and good activity versus 7-methylguanosine (30% that of the WT) [21]. Therefore, we decided to confirm the binding using other approaches, namely Absence of any conformational change upon P i binding to Arg24Ala mutant emphasizes the role of the Arg24 as a crucial residue for P i ligation in a stable, long-existing form (in the time scale of catalytic events).…”

Section: Phosphate Binding Is Affected By Arg24ala Mutation and Activmentioning

confidence: 99%

“…Enzymes were purified in a two-step procedure using Q-Sepharose FF anion exchange chromatography and gel filtration on Sephacryl S-200 column as previously described [21]. After purification, samples in 20 mM Tris-HCl, pH 7.4 were concentrated to 2.8 mM by ultrafiltration at 4000 g using Vivaspin 2 (10000 MWCO) centrifugal concentrators (Sartorius Stedim Biotech).…”

Section: E Coli Pnp Sample Preparationmentioning

confidence: 99%

“…This remarkable enzyme has been the subject of our scientific interest for many years [19][20][21][22][23][24]. PNPs are versatile catalysts of the reversible phosphorolysis of purine (2′-deoxy)-ribonucleosides [25,26]: purine (2′-deoxy)-ribonucleoside + orthophosphate ↔ purine base + (2′-deoxy)-ribose 1-phosphate Due to their catalytic function and much broader specificity compared with their trimeric human counterpart [26], hexameric PNPs (e.g., from E. coli) have been investigated for the efficient synthesis of nucleoside analogues [27] as well as for the activation of pro-drugs in anti-cancer gene therapies [28].…”

mentioning

confidence: 99%

“…This implies that a mechanism must exist for information transfer from the active site of one monomer to the other. However, no such mechanism could be derived from the E. coli PNP crystal structures [20,21]. The two active sites within a dimer are about 20 Å apart, and the flexible α-helices H8, one of which undergoes a conformational change, point in opposite directions.…”

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confidence: 99%

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New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations

Kazazić

Bertoša

Luić

et al. 2015

J. Am. Soc. Mass Spectrom.

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Abstract. The biologically active form of purine nucleoside phosphorylase (PNP) from Escherichia coli (EC 2.4.2.1) is a homohexamer unit, assembled as a trimer of dimers. Upon binding of phosphate, neighboring monomers adopt different active site conformations, described as open and closed. To get insight into the functions of the two distinctive active site conformations, virtually inactive Arg24Ala mutant is complexed with phosphate; all active sites are found to be in the open conformation. To understand how the sites of neighboring monomers communicate with each other, we have combined H/D exchange (H/DX) experiments with molecular dynamics (MD) simulations. Both methods point to the mobility of the enzyme, associated with a few flexible regions situated at the surface and within the dimer interface. Although H/ DX provides an average extent of deuterium uptake for all six hexamer active sites, it was able to indicate the dynamic mechanism of cross-talk between monomers, allostery. Using this technique, it was found that phosphate binding to the wild type (WT) causes arrest of the molecular motion in backbone fragments that are flexible in a ligand-free state. This was not the case for the Arg24Ala mutant. Upon nucleoside substrate/inhibitor binding, some release of the phosphate-induced arrest is observed for the WT, whereas the opposite effects occur for the Arg24Ala mutant. MD simulations confirmed that phosphate is bound tightly in the closed active sites of the WT; conversely, in the open conformation of the active site of the WT phosphate is bound loosely moving towards the exit of the active site. In Arg24Ala mutant binary complex P i is bound loosely, too.

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Section: Phosphate Binding Is Affected By Arg24ala Mutation and Activmentioning

confidence: 99%

Section: Phosphate Binding Is Affected By Arg24ala Mutation and Activmentioning

confidence: 99%

Section: E Coli Pnp Sample Preparationmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations

Kazazić

Bertoša

Luić

et al. 2015

J. Am. Soc. Mass Spectrom.

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Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base‐Modified Nucleosides

Fateev

Kharitonova

Antonov

et al. 2015

Chemistry A European J

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A wide range of natural purine analogues was used as probe to assess the mechanism of recognition by the wild-type (WT) E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant. The results were analyzed from viewpoint of the role of the Ser90 residue and the structural features of the bases. It was found that the Ser90 residue of the PNP 1) plays an important role in the binding and activation of 8-aza-7-deazapurines in the synthesis of their nucleosides, 2) participates in the binding of α-D-pentofuranose-1-phosphates at the catalytic site of the PNP, and 3) catalyzes the dephosphorylation of intermediary formed 2-deoxy-α-D-ribofuranose-1-phosphate in the trans-2-deoxyribosylation reaction. 5-Aza-7-deazaguanine manifested excellent substrate activity for both enzymes, 8-amino-7-thiaguanine and 2-aminobenzothiazole showed no substrate activity for both enzymes. On the contrary, the 2-amino derivatives of benzimidazole and benzoxazole are substrates and are converted into the N1- and unusual N2-glycosides, respectively. 9-Deaza-5-iodoxanthine showed moderate inhibitory activity of the WT E. coli PNP, whereas 9-deazaxanthine and its 2'-deoxyriboside are weak inhibitors.

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Unique substrate specificity of purine nucleoside phosphorylases from Thermus thermophilus

2013

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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.

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Validation of the catalytic mechanism of Escherichia coli purine nucleoside phosphorylase by structural and kinetic studies

Cited by 38 publications

References 23 publications

New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations

New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations

Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base‐Modified Nucleosides

Unique substrate specificity of purine nucleoside phosphorylases from Thermus thermophilus

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