The X-ray structure of<i>Salmonella typhimurium</i>uridine nucleoside phosphorylase complexed with 2,2′-anhydrouridine, phosphate and potassium ions at 1.86 Å resolution

Lashkov, A.A.; Жухлистова, Н. Е.; Gabdoulkhakov, A.G.; Shtil, Alexander A.; Efremov, Roman G.; Betzel, Christian; Mikhailov, A.M.

doi:10.1107/s0907444909044175

Cited by 21 publications

(14 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our understanding of the fundamental catalytic mechanisms underlying UPP activity was founded on the structural analysis of bacterial UPPs, first with E. coli UPP (Morgunova et al, 1995; Burling et al, 2003; Caradoc-Davies et al, 2004; Bu et al, 2005) followed by research on the closely-related S. typhimurium homologue (Dontsova et al, 2005; Lashkov et al, 2009, 2010). Only recently have multiple structures of the human enzyme, hUPP1 (Roosild et al, 2009; Roosild and Castronovo, 2010), its bovine homologue, bUPP1 (Paul et al, 2010), and a UPP from the parasitic protozoa, Trypanosoma brucei (Larson et al, 2010), revealed key differences between prokaryotic and eukaryotic variations of this enzyme.…”

Section: Introductionmentioning

confidence: 99%

A novel structural mechanism for redox regulation of uridine phosphorylase 2 activity

Roosild¹,

Castronovo²,

Villoso³

et al. 2011

Journal of Structural Biology

View full text Add to dashboard Cite

Uridine phosphorylase (UPP) catalyzes the reversible conversion of uridine to uracil and ribose-1-phosphate and plays an important pharmacological role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil and capecitabine. Most vertebrate animals, including humans, possess two homologues of this enzyme (UPP1 & UPP2), of which UPP1 has been more thoroughly studied and is better characterized. Here, we report two crystallographic structures of human UPP2 (hUPP2) in distinctly active and inactive conformations. These structures reveal that a conditional intramolecular disulfide bridge can form within the protein that dislocates a critical phosphate-coordinating arginine residue (R100) away from the active site, disabling the enzyme. In vitro activity measurements on both recombinant hUPP2 and native mouse UPP2 confirm the redox sensitivity of this enzyme, in contrast to UPP1. Sequence analysis shows that this feature is conserved among UPP2 homologues and lacking in all UPP1 proteins due to the absence of a necessary cysteine residue. The state of the disulfide bridge has further structural consequences for one face of the enzyme that suggest UPP2 may have additional functions in sensing and initiating cellular responses to oxidative stress. The molecular details surrounding these dynamic aspects of hUPP2 structure and regulation provide new insights as to how novel inhibitors of this protein may be developed with improved specificity and affinity. As uridine is emerging as a promising protective compound in neuro-degenerative diseases, including Alzheimer’s and Parkinson’s, understanding the regulatory mechanisms underlying UPP control of uridine concentration is key to improving clinical outcomes in these illnesses.

show abstract

Section: Introductionmentioning

confidence: 99%

A novel structural mechanism for redox regulation of uridine phosphorylase 2 activity

Roosild¹,

Castronovo²,

Villoso³

et al. 2011

Journal of Structural Biology

View full text Add to dashboard Cite

show abstract

“…These latter residues, which are key to binding the benzyl modification of high affinity inhibitors such as BAU, are also the only distinguishing active site residues when comparing eukaryotic and prokaryotic enzymes (equivalent E. coli residues are Ile 220, Val221 and P229). This is an important consideration when contemplating generating selectivity in such competitive inhibitors between the two enzymes, as would be needed for the development of effective antibiotics targeting only bacterial homologues of this protein [19].…”

Section: Resultsmentioning

confidence: 99%

“…A fundamental understanding of the underlying structural mechanisms behind the catalytic activity of this enzyme has been established through extensive structural analysis of bacterial UPPs, starting with E. coli UPP (EcUPP) [13]–[16] and then the closely-related S. typhimurium homologue [17]–[19]. More recently, multiple structures of the human enzyme, hUPP1 [20], its bovine homologue, bUPP1 [21], and a UPP from the parasitic protozoa, Trypanosoma brucei [22], have been determined.…”

Section: Introductionmentioning

confidence: 99%

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Roosild¹,

Castronovo²

2010

PLoS ONE

View full text Add to dashboard Cite

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications.

show abstract

“…The conformation of 2,2'-O-anhydrouridine is high-syn (χ = 111 • ) with P~230 • . This conformation is observed in all three active sites in complexes with Salmonella typhimurium UP (PDB 3FWP) [53] and it is determined by the C2'-O-C2 covalent bond. The rigid structure of 2,2'-O-anhydrouridine is well accommodated by the active site of uridine phosphorylase.…”

Section: Conformations Of Nucleosides and Their Analogues In The Actimentioning

confidence: 98%

Strained Conformations of Nucleosides in Active Sites of Nucleoside Phosphorylases

2020

View full text Add to dashboard Cite

Nucleoside phosphorylases catalyze the reversible phosphorolysis of nucleosides to heterocyclic bases, giving α-d-ribose-1-phosphate or α-d-2-deoxyribose-1-phosphate. These enzymes are involved in salvage pathways of nucleoside biosynthesis. The level of these enzymes is often elevated in tumors, which can be used as a marker for cancer diagnosis. This review presents the analysis of conformations of nucleosides and their analogues in complexes with nucleoside phosphorylases of the first (NP-1) family, which includes hexameric and trimeric purine nucleoside phosphorylases (EC 2.4.2.1), hexameric and trimeric 5′-deoxy-5′-methylthioadenosine phosphorylases (EC 2.4.2.28), and uridine phosphorylases (EC 2.4.2.3). Nucleosides adopt similar conformations in complexes, with these conformations being significantly different from those of free nucleosides. In complexes, pentofuranose rings of all nucleosides are at the W region of the pseudorotation cycle that corresponds to the energy barrier to the N↔S interconversion. In most of the complexes, the orientation of the bases with respect to the ribose is in the high-syn region in the immediate vicinity of the barrier to syn ↔ anti transitions. Such conformations of nucleosides in complexes are unfavorable when compared to free nucleosides and they are stabilized by interactions with the enzyme. The sulfate (or phosphate) ion in the active site of the complexes influences the conformation of the furanose ring. The binding of nucleosides in strained conformations is a characteristic feature of the enzyme–substrate complex formation for this enzyme group.

show abstract

The X-ray structure ofSalmonella typhimuriumuridine nucleoside phosphorylase complexed with 2,2′-anhydrouridine, phosphate and potassium ions at 1.86 Å resolution

Cited by 21 publications

References 39 publications

A novel structural mechanism for redox regulation of uridine phosphorylase 2 activity

A novel structural mechanism for redox regulation of uridine phosphorylase 2 activity

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Strained Conformations of Nucleosides in Active Sites of Nucleoside Phosphorylases

Contact Info

Product

Resources

About