High-synconformation of uridine and asymmetry of the hexameric molecule revealed in the high-resolution structures ofShewanella oneidensisMR-1 uridine phosphorylase in the free form and in complex with uridine

2014 ◽  
Vol 70 (12) ◽  
pp. 3310-3319 ◽  
Author(s):  
Tatyana N. Safonova ◽  
Sergey N. Mikhailov ◽  
Vladimir P. Veiko ◽  
Nadezhda N. Mordkovich ◽  
Valentin A. Manuvera ◽  
...  
Keyword(s):  
High Resolution ◽  
Active Sites ◽  
Shewanella Oneidensis ◽  
Free Form ◽  
Uridine Phosphorylase ◽  
Glycerol Molecule ◽  
Closed Conformation ◽  
Pyrimidine Salvage ◽  
Key Enzyme ◽  
Crystallization Solution

Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Expression of UP fromShewanella oneidensisMR-1 (SoUP) was performed inEscherichia coli. The high-resolution X-ray structure of SoUP was solved in the free form and in complex with uridine. A crystal of SoUP in the free form was grown under microgravity and diffracted to ultrahigh resolution. Both forms of SoUP contained sulfate instead of phosphate in the active site owing to the presence of ammonium sulfate in the crystallization solution. The latter can be considered as a good mimic of phosphate. In the complex, uridine adopts a high-synconformation with a nearly planar ribose ring and is present only in one subunit of the hexamer. A comparison of the structures of SoUP in the free form and in complex with the natural substrate uridine showed that the subunits of the hexamer are not identical, with the active sites having either an open or a closed conformation. In the monomers with the closed conformation, the active sites in which uridine is absent contain a glycerol molecule mimicking the ribose moiety of uridine.

Concerted action of two subunits of the functional dimer ofShewanella oneidensisMR-1 uridine phosphorylase derived from a comparison of the C212S mutant and the wild-type enzyme

2016 ◽  
Vol 72 (2) ◽  
pp. 203-210 ◽  
Author(s):  
T. N. Safonova ◽  
N. N. Mordkovich ◽  
V. P. Veiko ◽  
N. A. Okorokova ◽  
V. A. Manuvera ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Shewanella Oneidensis ◽  
Dimensional Structure ◽  
Free Form ◽  
Uridine Phosphorylase ◽  
Wild Type ◽  
Concerted Action ◽  
Wild Type Enzyme ◽  
Pyrimidine Salvage ◽  
Key Enzyme

Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The structure of the C212S mutant of uridine phosphorylase from the facultatively aerobic Gram-negative γ-proteobacteriumShewanella oneidensisMR-1 (SoUP) was determined at 1.68 Å resolution. A comparison of the structures of the mutant and the wild-type enzyme showed that one dimer in the mutant hexamer differs from all other dimers in the mutant and wild-type SoUP (both in the free form and in complex with uridine). The key difference is the `maximum open' state of one of the subunits comprising this dimer, which has not been observed previously for uridine phosphorylases. Some conformational features of the SoUP dimer that provide access of the substrate into the active site are revealed. The binding of the substrate was shown to require the concerted action of two subunits of the dimer. The changes in the three-dimensional structure induced by the C212S mutation account for the lower affinity of the mutant for inorganic phosphate, while the affinity for uridine remains unchanged.

scholarly journals Crystallization of uridine phosphorylase fromShewanella oneidensisMR-1 in the laboratory and under microgravity and preliminary X-ray diffraction analysis

2012 ◽  
Vol 68 (11) ◽  
pp. 1387-1389 ◽  
Author(s):  
Tatyana N. Safonova ◽  
Nadezhda N. Mordkovich ◽  
Konstantin M. Polyakov ◽  
Valentin A. Manuvera ◽  
Vladimir P. Veiko ◽  
...  
Keyword(s):  
Space Station ◽  
Shewanella Oneidensis ◽  
Free Form ◽  
Diffusion Method ◽  
Data Sets ◽  
Uridine Phosphorylase ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Key Enzyme

Uridine phosphorylase (UDP, EC 2.4.2.3), a key enzyme in the pyrimidine salvage pathway, catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The gene expression of UDP fromShewanella oneidensisMR-1 was performed in the recipient strainEscherichia coli. The UDP protein was crystallized on earth (in the free form and in complex with uridine as the substrate) by the hanging-drop vapour-diffusion method at 296 K and under microgravity conditions (in the free form) aboard the Russian Segment of the International Space Station by the capillary counter-diffusion method. The data sets were collected to a resolution of 1.9 Å from crystals of the free form grown on earth, 1.6 Å from crystals of the complex with uridine and 0.95 Å from crystals of the free form grown under microgravity. All crystals belong to the space groupP21and have similar unit-cell parameters. The crystal of uridine phosphorylase grown under microgravity diffracted to ultra-high resolution and gave high-quality X-ray diffraction data.

Determination of the GH3.12 protein conformation through HPLC-integrated SAXS measurements combined with X-ray crystallography

2013 ◽  
Vol 69 (10) ◽  
pp. 2072-2080 ◽  
Author(s):  
Adam Round ◽  
Elizabeth Brown ◽  
Romain Marcellin ◽  
Ulrike Kapp ◽  
Corey S. Westfall ◽  
...  
Keyword(s):  
Protein Conformation ◽  
Active Sites ◽  
Solution Structure ◽  
Critical Role ◽  
X Ray ◽  
Hplc Purification ◽  
Closed Conformation ◽  
Terminal Domain ◽  
Redundant Data ◽  
On Line

The combination of protein crystallography and small-angle X-ray scattering (SAXS) provides a powerful method to investigate changes in protein conformation. These complementary structural techniques were used to probe the solution structure of the apo and the ligand-bound forms of theArabidopsis thalianaacyl acid–amido synthetase GH3.12. This enzyme is part of the extensive GH3 family and plays a critical role in the regulation of plant hormones through the formation of amino-acid-conjugated hormone productsviaan ATP-dependent reaction mechanism. The enzyme adopts two distinct C-terminal domain orientations with `open' and `closed' active sites. Previous studies suggested that ATP only binds in the open orientation. Here, the X-ray crystal structure of GH3.12 is presented in the closed conformation in complex with the nonhydrolysable ATP analogue AMPCPP and the substrate salicylate. Using on-line HPLC purification combined with SAXS measurements, the most likely apo and ATP-bound protein conformations in solution were determined. These studies demonstrate that the C-terminal domain is flexible in the apo form and favours the closed conformation upon ATP binding. In addition, these data illustrate the efficacy of on-line HPLC purification integrated into the SAXS sample-handling environment to reliably monitor small changes in protein conformation through the collection of aggregate-free and highly redundant data.

Structural study of the X-ray-induced enzymatic reaction of octahaem cytochromecnitrite reductase

2015 ◽  
Vol 71 (5) ◽  
pp. 1087-1094 ◽  
Author(s):  
A. A. Trofimov ◽  
K. M. Polyakov ◽  
V. A. Lazarenko ◽  
A. N. Popov ◽  
T. V. Tikhonova ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Catalytic Reaction ◽  
Active Sites ◽  
Enzymatic Reaction ◽  
Absorbed Dose ◽  
Free Form ◽  
Data Sets ◽  
X Rays ◽  
X Ray ◽  
Site Water

Octahaem cytochromecnitrite reductase from the bacteriumThioalkalivibrio nitratireducenscatalyzes the reduction of nitrite to ammonium and of sulfite to sulfide. The reducing properties of X-ray radiation and the high quality of the enzyme crystals allow study of the catalytic reaction of cytochromecnitrite reductase directly in a crystal of the enzyme, with the reaction being induced by X-rays. Series of diffraction data sets with increasing absorbed dose were collected from crystals of the free form of the enzyme and its complexes with nitrite and sulfite. The corresponding structures revealed gradual changes associated with the reduction of the catalytic haems by X-rays. In the case of the nitrite complex the conversion of the nitrite ions bound in the active sites to NO species was observed, which is the beginning of the catalytic reaction. For the free form, an increase in the distance between the oxygen ligand bound to the catalytic haem and the iron ion of the haem took place. In the case of the sulfite complex no enzymatic reaction was detected, but there were changes in the arrangement of the active-site water molecules that were presumably associated with a change in the protonation state of the sulfite ions.

scholarly journals Psoromic Acid, a Lichen-Derived Molecule, Inhibits the Replication of HSV-1 and HSV-2, and Inactivates HSV-1 DNA Polymerase: Shedding Light on Antiherpetic Properties

Molecules ◽  
2019 ◽  
Vol 24 (16) ◽  
pp. 2912 ◽  
Author(s):  
Sherif T. S. Hassan ◽  
Miroslava Šudomová ◽  
Kateřina Berchová-Bímová ◽  
Karel Šmejkal ◽  
Javier Echeverría
Keyword(s):  
Dna Polymerase ◽  
Active Sites ◽  
Inhibitory Action ◽  
Inhibitory Concentration ◽  
Competitive Inhibition ◽  
Inhibitory Effect ◽  
Standard Drug ◽  
Key Enzyme ◽  
Hsv 1

Psoromic acid (PA), a bioactive lichen-derived compound, was investigated for its inhibitory properties against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), along with the inhibitory effect on HSV-1 DNA polymerase, which is a key enzyme that plays an essential role in HSV-1 replication cycle. PA was found to notably inhibit HSV-1 replication (50% inhibitory concentration (IC50): 1.9 μM; selectivity index (SI): 163.2) compared with the standard drug acyclovir (ACV) (IC50: 2.6 μM; SI: 119.2). The combination of PA with ACV has led to potent inhibitory activity against HSV-1 replication (IC50: 1.1 µM; SI: 281.8) compared with that of ACV. Moreover, PA displayed equivalent inhibitory action against HSV-2 replication (50% effective concentration (EC50): 2.7 μM; SI: 114.8) compared with that of ACV (EC50: 2.8 μM; SI: 110.7). The inhibition potency of PA in combination with ACV against HSV-2 replication was also detected (EC50: 1.8 µM; SI: 172.2). Further, PA was observed to effectively inhibit HSV-1 DNA polymerase (as a non-nucleoside inhibitor) with respect to dTTP incorporation in a competitive inhibition mode (half maximal inhibitory concentration (IC50): 0.7 μM; inhibition constant (Ki): 0.3 μM) compared with reference drugs aphidicolin (IC50: 0.8 μM; Ki: 0.4 μM) and ACV triphosphate (ACV-TP) (IC50: 0.9 μM; Ki: 0.5 μM). It is noteworthy that the mechanism by which PA-induced anti-HSV-1 activity was related to its inhibitory action against HSV-1 DNA polymerase. Furthermore, the outcomes of in vitro experiments were authenticated using molecular docking analyses, as the molecular interactions of PA with the active sites of HSV-1 DNA polymerase and HSV-2 protease (an essential enzyme required for HSV-2 replication) were revealed. Since this is a first report on the above-mentioned properties, we can conclude that PA might be a future drug for the treatment of HSV infections as well as a promising lead molecule for further anti-HSV drug design.

scholarly journals Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Catherine P. Chia ◽  
Noriko Inoguchi ◽  
Kyle C. Varon ◽  
Bradley M. Bartholomai ◽  
Hideaki Moriyama
Keyword(s):  
Dictyostelium Discoideum ◽  
Active Sites ◽  
Fluorescent Protein ◽  
Subcellular Fractionation ◽  
Unicellular Eukaryote ◽  
Gene Encoding ◽  
Conserved Motifs ◽  
N Terminus ◽  
Key Enzyme ◽  
Green Fluorescent

Abstract Objective The nuclear and mitochondrial genomes of Dictyostelium discoideum, a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectively. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. Results The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the shortened N-termini that arose from the loss of residues 1-37. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.

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