Structural Basis for Substrate Specificity of Escherichia coli Purine Nucleoside Phosphorylase

Bennett, Eric M.; Li, Chenglong; Allan, Paula W.; Parker, William B.; Ealick, S.E.

doi:10.1074/jbc.m304622200

Cited by 88 publications

(130 citation statements)

References 51 publications

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“…The PfPNP sequence has greater similarity to E. coli than to human PNP (9), and its structure is hexameric as is the E. coli enzyme (7,20,21). PfPNP, however, has unique substrate specificity when compared with either E. coli or mammalian PNPs (9,21).…”

Section: Resultsmentioning

confidence: 99%

“…PfPNP, however, has unique substrate specificity when compared with either E. coli or mammalian PNPs (9,21). PfPNP is specific for 6-oxopurine rings in its substrates, nucleosides with adenine rings (including MTA) being neither substrates nor inhibitors for the enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…We overexpressed and tested E. coli PNP, which has been extensively characterized structurally and enzymatically (20,21). E. coli PNP has activity against inosine and adenosine but has no detectable activity toward MTA or MTI.…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

Ting¹,

Shi

Lewandowicz

et al. 2005

Journal of Biological Chemistry

111

182

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Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of polyamine synthesis are recycled in a novel pathway in which 5 -methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5 -methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5 -methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not been described previously. 5 -Methylthio-immucillin-H, a transition state analogue inhibitor that is selective for malarial relative to human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may also have application as anti-malarials.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

Ting¹,

Shi

Lewandowicz

et al. 2005

Journal of Biological Chemistry

111

182

View full text Add to dashboard Cite

show abstract

“…The complex crystallized in a hexagonal space group P6 1 22 with only half of the hexamer in a asym- The active sites of monomers B and C ( Figure 6) contain two phosphate molecules each. Interestingly, none of these ions make previously observed hydrogen bonds 7,9,13,20,21 to the nucleoside and residues Gly20, Arg87, Ser90 and Arg43 from the neighbouring monomer. These two ions make hydrogen bonds to each other while the "standard" phosphate position is occupied by three water molecules.…”

Section: Ternary Complex Of E Coli Pnp With Formycin a And Phosphatementioning

confidence: 99%

Still a Long Way to Fully Understanding the Molecular Mechanism of Escherichia coli Purine Nucleoside Phosphorylase

et al. 2013

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Abstract. The results of several decades of studying the catalytic mechanism of Escherichia coli purine nucleoside phosphorylases (PNP) by solution studies and crystal structure determinations are presented. Potentially PNPs can be used for enzyme-activating prodrug gene therapy against solid tumours because of the differences in specificity between human and E. coli PNPs. Biologically active form of PNP from E. coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. Two conformations of the active site are possible after substrate(s) binding: open and closed. A series of determined 3D-structures of PNP binary and ternary complexes facilitated the prediction of the main steps in the catalytic mechanism. For their validation the active site mutants: Arg24Ala, Asp204Ala, Arg217Ala, Asp204Asn and double mutant Asp204Ala/Arg217Ala were prepared. The activity tests confirm that catalysis involves protonation of the purine base at position N7 and give better insight into the cooperativity between subunits in this oligomeric enzyme. (doi: 10.5562/cca2116)

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“…Previously, E. coli PNP was shown to be active in mice (47). However, substrate specificities differ among PNPs of different species (48). Nevertheless, because of the greater than 85% amino acid homology between human and murine PNP, we anticipated that human PNP would also be active in mice.…”

Section: Figurementioning

confidence: 99%

TAT-mediated intracellular delivery of purine nucleoside phosphorylase corrects its deficiency in mice

Toro-Montoya¹,

Grunebaum²

2006

Journal of Clinical Investigation

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Defects in purine nucleoside phosphorylase (PNP) enzyme activity result in abnormal nucleoside homeostasis, severe T cell immunodeficiency, neurological dysfunction, and early death. Protein transduction domain (PTD) can transfer molecules into cells and may help restore PNP activity in cases of PNP deficiency. However, long-term use of PTD to replace enzymes in animal models or patients has not previously been described. We fused human PNP to the HIV-TAT PTD and found that the fusion with TAT changed the retention and distribution of PNP in PNP-deficient mice. TAT induced rapid intracellular delivery of PNP into tissues, including the brain, prevented urinary excretion of PNP, and protected PNP from neutralizing antibodies, resulting in significant extension of the enzyme's biological activity in vivo. Frequent TAT-PNP injections in PNP-deficient mice corrected the metabolic disorder and immune defects with no apparent toxicity. TAT-PNP remained effective over 24 weeks of treatment, resulting in continued improvement in immune function and extended survival. Our data demonstrate that TAT changes the properties of PNP in vivo and that long-term intracellular delivery of PNP by TAT corrects PNP deficiency in mice. We provide evidence to promote further use of PTD to treat diseases that require repeated intracellular enzyme or protein delivery.

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Structural Basis for Substrate Specificity of Escherichia coli Purine Nucleoside Phosphorylase

Cited by 88 publications

References 51 publications

Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

Still a Long Way to Fully Understanding the Molecular Mechanism of Escherichia coli Purine Nucleoside Phosphorylase

TAT-mediated intracellular delivery of purine nucleoside phosphorylase corrects its deficiency in mice

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