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Literature summary for 2.4.2.1 extracted from

  • Stefanic, Z.; Mikleusevic, G.; Luic, M.; Bzowska, A.; Lescic Asler, I.
    Structural characterization of purine nucleoside phosphorylase from human pathogen Helicobacter pylori (2017), Int. J. Biol. Macromol., 101, 518-526 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene deoD, DNA and amino acid sequence determination and analysis of clinical isolate PNP-Zg encoded by a deoD gene, sequence comparisons, recombinant expression of PNP-Zg in Escherichia coli strain BL21-CodonPlus(DE3)-RIL Helicobacter pylori

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant enzyme PNP-Zg in ternary complex with hypoxanthine and phosphate molecules, obtained from three different crystallization conditions: B8 (0.2 M MgCl2, 0.1 M Tris-HCl, pH 7.0, 10 % PEG 8000 for structure HpPNP-1), A1 (0.2 M Li2SO4, 0.1 M Na acetate, pH 4.5, 50 % PEG 400 for structure HpPNP-2) and D3 (0.2 M Na chloride, 0.1 M Na/K phosphate, pH 6.2, 50% PEG 200 for structure HpPNP-3), 20°C, X-ray diffraction structure determination and analysis at 2.3-2.4 A resolution, molecular replacement using the structure of Escherichia coli PNP (PDB ID 1K9S) as template Helicobacter pylori

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
142000
-
recombinant enzyme, gel filtration Helicobacter pylori
154716
-
sequence comparison Helicobacter pylori

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
7-methylguanosine + phosphate Helicobacter pylori low activity 7-methylguanine + alpha-D-ribose 1-phosphate
-
r
adenosine + phosphate Helicobacter pylori very low activity adenine + alpha-D-ribose 1-phosphate
-
r
guanosine + phosphate Helicobacter pylori
-
guanine + alpha-D-ribose 1-phosphate
-
r
inosine + phosphate Helicobacter pylori
-
hypoxanthine + alpha-D-ribose 1-phosphate
-
r

Organism

Organism UniProt Comment Textmining
Helicobacter pylori A0A0G2R4X7 multifunctional fusion protein; clinical isolate PNP-Zg
-

Purification (Commentary)

Purification (Comment) Organism
recombinant enzyme PNP-Zg from Escherichia coli strain BL21-CodonPlus(DE3)-RIL by formycin affinity chromatography (elution with inosine), dialysis, and gel filtration, to homogeneity Helicobacter pylori

Reaction

Reaction Comment Organism Reaction ID
purine ribonucleoside + phosphate = purine + alpha-D-ribose 1-phosphate molecular reaction mechanism, overview Helicobacter pylori

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.2
-
purified recombinant enzyme, pH 7.0, 25°C, substrate adenosine Helicobacter pylori
7.5
-
purified recombinant enzyme, pH 7.0, 25°C, substrate 7-methylguanosine Helicobacter pylori
80.4
-
purified recombinant enzyme, pH 7.0, 25°C, substrate inosine Helicobacter pylori

Storage Stability

Storage Stability Organism
storage of the purified enzyme at 4°C for one month at 0.4 mg/ml in 100 mM Tris-HCl, pH 7.6, results in an about 50% decrease in activity Helicobacter pylori

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
7-methylguanosine + phosphate low activity Helicobacter pylori 7-methylguanine + alpha-D-ribose 1-phosphate
-
r
adenosine + phosphate very low activity Helicobacter pylori adenine + alpha-D-ribose 1-phosphate
-
r
guanosine + phosphate
-
Helicobacter pylori guanine + alpha-D-ribose 1-phosphate
-
r
inosine + phosphate
-
Helicobacter pylori hypoxanthine + alpha-D-ribose 1-phosphate
-
r

Subunits

Subunits Comment Organism
homohexamer 6 * 24900, recombinant enzyme, SDS-PAGE, 6 * 25786, sequence calculation Helicobacter pylori
More each monomer requires a portion of other monomer for the completion of the active site. The two monomers connected by approximate two-fold symmetry in this way form a dimer, where the monomers mutually donate to the active site amino acids His4 and Arg43. Such dimers are repeated by an approximate three-fold axis that is perpendicular to the two-fold axis to complete a set of three dimers that make the whole homohexamer of PNP, secondary structure analysis, overview Helicobacter pylori

Synonyms

Synonyms Comment Organism
DeoD
-
Helicobacter pylori
EC592_06680 locus name Helicobacter pylori
HpPNP
-
Helicobacter pylori
PNP
-
Helicobacter pylori
PNP-Zg
-
Helicobacter pylori
purine nucleoside orthophosphate ribosyl transferase
-
Helicobacter pylori
purine nucleoside phosphorylase
-
Helicobacter pylori

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Helicobacter pylori

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
20 80 purified recombinant enzyme, pH 7.0, 1 h. The enzyme is stable retaining over 90% of the initial activity at up to 37°C at pH 7.0, the activity is completely abolished at 60°C Helicobacter pylori

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
assay at Helicobacter pylori

pH Stability

pH Stability pH Stability Maximum Comment Organism
2 12 purified recombinant enzyme, 25°C, 1 h. The enzyme is stable retaining over 90% of the initial activity in the pH range 6.0-9.0 at 25°C. Stability decreases rapidly below pH 5.5 and above pH 9.5 Helicobacter pylori

General Information

General Information Comment Organism
metabolism the purine metabolism in Helicobacter pylori is solely dependant on the salvage pathway and one of the key enzymes in this pathway is purine nucleoside phosphorylase (PNP) Helicobacter pylori
additional information the structure of Helicobacter pylori PNP is typical for high molecular mass PNPs. Active site structure and ligand binding structure analysis, overview. The structure of Helicobacter pylori PNP-Zg represents the first reported case of a true dead-end complex of bacterial hexameric PNP bound with one of the reaction substrates, phosphate, and one of the reaction products, hypoxanthine, which is produced by the enzyme turnover during the purification procedure Helicobacter pylori
physiological function PNP is the key enzyme in the purine salvage pathway. It catalyses the reversible phosphorolytic cleavage of the glycosidic bond of purine nucleosides (and some analogues) in the presence of phosphate as a second substrate Helicobacter pylori