Crystal structure of calf spleen purine nucleoside phosphorylase complexed to a novel purine analogue

Pereira, H.M.; Berdini, Valério; Cleasby, Anne; Garratt, R.C.

doi:10.1016/j.febslet.2007.09.051

Cited by 9 publications

(10 citation statements)

References 25 publications

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“…The optimum pH of Klac PNP is comparable to the optimum pH values reported for human, calf spleen, and S . cerevisiae PNPs: 7.0, 7.0, and 7.5, respectively [ 43 , 48 , 68 ]. Higher molecular weight hexameric PNPs of prokaryotic origin are in general more thermostable, as compared to the low molecular weight trimeric PNPs [ 41 ].…”

Section: Resultsmentioning

confidence: 99%

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food

et al. 2016

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Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a combination of engineered KlacPNP and other enzymes involved in purine degradation could effectively lower the purine content in foods and beverages.

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Section: Resultsmentioning

confidence: 99%

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food

et al. 2016

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“…A paper by Pereira et al has an interesting twist beyond the identification of a binder [23]. They were searching for inhibitors of purine nucleoside phosphorylase (PNP) using the program GOLD.…”

Section: Complete Studiesmentioning

confidence: 99%

“…The interaction energies of the low-energy consensus configurations from families one and two are −11.2 and −3.2 kcal.mol, respectively. Energies were calculated with Chemgrid (23) with the use of the AMBER potential (32). The maximum van der Waals energy for any atom was set to 10 kcal/mol with a 10 Å distance cutoff in a 28 Å box centered on the SB atoms.…”

Section: Figurementioning

confidence: 99%

Docking Screens: Right for the Right Reasons?

Kolb¹,

Irwin²

2009

CTMC

116

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Whereas docking screens have emerged as the most practical way to use protein structure for ligand discovery, an inconsistent track record raises questions about how well docking actually works. In its favor, a growing number of publications report the successful discovery of new ligands, often supported by experimental affinity data and controls for artifacts. Few reports, however, actually test the underlying structural hypotheses that docking makes. To be successful and not just lucky, prospective docking must not only rank a true ligand among the top scoring compounds, it must also correctly orient the ligand so the score it receives is biophysically sound. If the correct binding pose is not predicted, a skeptic might well infer that the discovery was serendipitous. Surveying over 15 years of the docking literature, we were surprised to discover how rarely sufficient evidence is presented to establish whether docking actually worked for the right reasons. The paucity of experimental tests of theoretically predicted poses undermines confidence in a technique that has otherwise become widely accepted. Of course, solving a crystal structure is not always possible, and even when it is, it can be a lot of work, and is not readily accessible to all groups. Even when a structure can be determined, investigators may prefer to gloss over an erroneous structural prediction to better focus on their discovery. Still, the absence of a direct test of theory by experiment is a loss for method developers seeking to understand and improve docking methods. We hope this review will motivate investigators to solve structures and compare them with their predictions whenever possible, to advance the field.

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“…1,4,6 Structure-based approaches have become vital components in the design of enzyme inhibitors and receptor ligands. 11,14 Previously, we have crystallized SmPNP and determined its threedimensional structure in complex with different ligands. 6 As part of our ongoing research program aimed at discovering selective inhibitors of the parasite enzyme, we have carried out kinetic and crystallographic studies on a series of deazaguanines and other modified purine bases.…”

Section: Introductionmentioning

confidence: 99%

Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: Kinetic and structural studies

Castilho

Postigo

Pereira

et al. 2010

Bioorganic & Medicinal Chemistry

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Crystal structure of calf spleen purine nucleoside phosphorylase complexed to a novel purine analogue

Cited by 9 publications

References 25 publications

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food

Docking Screens: Right for the Right Reasons?

Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: Kinetic and structural studies

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