Open and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism

Koellner, G.; Bzowska, Agnieszka; Wielgus‐Kutrowska, Beata; Luić, Marija; Steiner, Thomas; Saenger, Wolfram; Stępiński, Janusz

doi:10.1006/jmbi.2001.5211

Cited by 77 publications

(150 citation statements)

References 50 publications

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“…Ligand-free systems were produced from 1ECP (PDB code) PNP crystal structure [50], in the apo form with all six sites in the open conformation; all WT and Arg24Ala mutant complexes were built from 1K9S (PDB code) WT PNP crystal structure [20], which has three active sites in open and three in closed conformations. Sulphate ions that are present in this structure were replaced by P i .…”

Section: Molecular Dynamics Simulationsmentioning

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.…”

mentioning

confidence: 99%

“…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. The interface of subunits forming a dimer buries 32 water molecules that are not connected with the bulk water [20]. An intriguing question is how the information on the conformational changes of one active site is transferred to the neighboring subunit within the dimer.…”

mentioning

confidence: 99%

“…In the present work, MD simulations were carried out to provide a dynamic view of the protein ground state within a solvent environment for all experimentally investigated protein forms. These structures represent a better comparison basis for the interpretation of the H/DX data than a single static X-ray structure of the ternary complex published earlier [20].…”

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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: Molecular Dynamics Simulationsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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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Formycins and their Analogues: Purine Nucleoside Phosphorylase Inhibitors and their Potential Application in Immunosuppression and Cancer

Bzowska

2008

Modified Nucleosides

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Production, Characterization and Synthetic Application of a Purine Nucleoside Phosphorylase from Aeromonas hydrophila

Ubiali

Serra

et al. 2012

Adv Synth Catal

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Purine nucleoside phosphorylase (PNP) from Aeromonas hydrophila encoded by the deoD gene has been over-expressed in Escherichia coli, purified, characterized about its substrate specificity and used for the preparative synthesis of some 6-substituted purine-9-ribosides. Substrate specificity towards natural nucleosides showed that this PNP catalyzes the phosphorolysis of both 6-oxo-and 6-aminopurine (deoxy)ribonucleosides. A library of nucleoside analogues was synthesized and then submitted to enzymatic phosphorolysis as well. This assay revealed that 1-, 2-, 6-and 7-modified nucleosides are accepted as substrates, whereas 8-substituted nucleosides are not. A few transglycosylation reactions were carried out using 7-methylguanosine iodide (4) as a d-ribose donor and 6-substituted purines as acceptor. In particular, following this approach, 2-amino-6-chloropurine-9-riboside (2c), 6-methoxypurine-9-riboside (2d) and 2-amino-6-(methylthio)purine-9-riboside (2g) were synthesized in very high yield and purity.

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Open and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism

Cited by 77 publications

References 50 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

Formycins and their Analogues: Purine Nucleoside Phosphorylase Inhibitors and their Potential Application in Immunosuppression and Cancer

Production, Characterization and Synthetic Application of a Purine Nucleoside Phosphorylase from Aeromonas hydrophila

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