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Literature summary for 3.6.1.1 extracted from

  • Jamwal, A.; Yogavel, M.; Abdin, M.Z.; Jain, S.K.; Sharma, A.
    Structural and biochemical characterization of apicomplexan inorganic pyrophosphatases (2017), Sci. Rep., 7, 5255 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.6.1.1

Cloned(Commentary)

Cloned (Comment) Organism
quantitative RT-PCR expression analysis, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21 Toxoplasma gondii
quantitative RT-PCR expression analysis, recombinant expression of MBP-His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 Plasmodium falciparum

Crystallization (Commentary)

Crystallization (Comment) Organism
purified enzyme, vapour diffusion method, mixing of 10 mg/ml protein solution with 10% PEG 4000, 20% glycerol, 0.03 M glycols, and 0.1 M HEPES/MOPS, pH 7.5, 20°C, 6-8 days, X-ray diffraction structure determination and analysis at 2.35 A resolution, molecular replacement method using ScPPase (PDB ID 1WGJ) as a template Toxoplasma gondii
purified recombinant detagged wild-type enzyme complexed with Mg2+, vapor diffusion method, 10 mg/ml seleno-methionine substituted PfPPase in buffer are mixed with condition 8% Tacsimate, pH 8.0, and 20% PEG 3350, 20°C, 4 days, X-ray diffraction structure determination and analysis at 2.35 A resolution Plasmodium falciparum

Protein Variants

Protein Variants Comment Organism
D198N site-directed mutagenesis, the mutant shows 5fold reduced kcat compared to wild-type Plasmodium falciparum
D203N site-directed mutagenesis, the mutant shows 600fold reduced activity compared to wild-type Plasmodium falciparum
D235N site-directed mutagenesis, the mutant shows 6fold reduced kcat compared to wild-type Plasmodium falciparum
K136R site-directed mutagenesis, the mutant shows a 40fold increased Km for diphosphate compared to wild-type Plasmodium falciparum
R158K site-directed mutagenesis, the mutant shows a moderately altered kcat and Km for diphosphate compared to wild-type Plasmodium falciparum

Inhibitors

Inhibitors Comment Organism Structure
NaF
-
 Plasmodium falciparum
NaF
-
 Toxoplasma gondii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.01
-
 diphosphate pH 7.2, 37°C, recombinant wild-type enzyme Plasmodium falciparum
0.0426
-
 diphosphate pH 7.2, 37°C, recombinant mutant D198N Plasmodium falciparum
0.0457
-
 diphosphate pH 7.2, 37°C, recombinant mutant D235N Plasmodium falciparum
0.0538
-
 diphosphate pH 7.2, 37°C, recombinant mutant D203N Plasmodium falciparum
0.064
-
 ATP pH 7.2, 37°C, recombinant enzyme Plasmodium falciparum
0.0642
-
 diphosphate pH 7.2, 37°C, recombinant mutant R158K Plasmodium falciparum
0.4028
-
 diphosphate pH 7.2, 37°C, recombinant mutant K136R Plasmodium falciparum

Localization

Localization Comment Organism GeneOntology No. Textmining
cytosol
-
 Plasmodium falciparum 5829
-
additional information PfPPase is not localized either in the apicoplast or the mitochondria in asexual parasite stages Plasmodium falciparum
-
-

Metals/Ions

Metals/Ions Comment Organism Structure
Co2+ stimulates Plasmodium falciparum
Mg2+ required Plasmodium falciparum
Mg2+ required, the active site of TgPPase contains two bound Mg2+ ions. One Mg2+ is bound at M1 site coordinated by Asp190, Asp195, and Asp227. The Mg2+ is bound to protein in M2 site predominantly through water molecules and Asp195. The observed conformation in the active site represents a state where both phosphates have already dissociated Toxoplasma gondii
Mn2+ stimulates Plasmodium falciparum
additional information the enzyme activity depends on Mg2+. Other divalent cations such as Co2+, Zn2+ and Mn2+ stimulate diphosphate hydrolysis but with lower efficiency. The relative PfPPase diphosphate activity confers by divalent metal ions fell in the descending order Mg2+, Co2+ and Zn2+, Mn2+. But Zn2+ is the preferred cofactor for hydrolysis of polyP3 and ATP Plasmodium falciparum
Zn2+ stimulates, Zn2+ is the preferred co-factor for hydrolysis of polyP3 and ATP Plasmodium falciparum

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
diphosphate + H2O Toxoplasma gondii
-
 2 phosphate
-
 ?
diphosphate + H2O Plasmodium falciparum
-
 2 phosphate
-
 ?

Organism

Organism UniProt Comment Textmining
Plasmodium falciparum C0H477
-
-
Toxoplasma gondii
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged enzyme from Escherichia coli strain BL21 by nickel affinity chromatography, anion exchange chromatography, and gel filtration Toxoplasma gondii
recombinant MBP-His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 by amylose affinity and nickel affinity chromatography, followed by TEV protease cleavage of both tags, dialysis, anion exchange chromatography, and ultrafiltration Plasmodium falciparum

Source Tissue

Source Tissue Comment Organism Textmining
additional information PfPPase is a cytosolic enzyme whose gene expression is upregulated during parasite asexual stages. Its expression increased relative to ring stage, during late trophozoite/early schizont and reduced again when schizonts mature. Protein expression occurs during all three stages but with higher expression during the trophozoite stage Plasmodium falciparum
-
schizont
-
 Plasmodium falciparum
-
trophozoite
-
 Plasmodium falciparum
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + H2O
-
 Plasmodium falciparum ?
-
 ?
diphosphate + H2O
-
 Toxoplasma gondii 2 phosphate
-
 ?
diphosphate + H2O
-
 Plasmodium falciparum 2 phosphate
-
 ?
additional information PfPPase is capable of utilizing PPi, polyP3, and ATP as substrates Plasmodium falciparum ?
-
-

Subunits

Subunits Comment Organism
dimer 2 * 45000, recombinant enzyme, SDS-PAGE Plasmodium falciparum
dimer TgPPase forms dimers in solution and in the crystal. Structure comparisons of the enzyme from Plasmodium falciparum (PfPPase) and Toxoplasma gondii (TgPPase), overview Toxoplasma gondii
More enzyme PfPPase exists predominantly as a dimer in solution with a minor tetrameric peak. PfPPase low complexity asparagine-rich N-terminal region mediates its dimerization. Structure comparisons of the enzyme from Plasmodium falciparum (PfPPase) and Toxoplasma gondii (TgPPase), overview Plasmodium falciparum

Synonyms

Synonyms Comment Organism
inorganic PPase
-
 Toxoplasma gondii
inorganic PPase
-
 Plasmodium falciparum
inorganic pyrophosphatase
-
 Toxoplasma gondii
inorganic pyrophosphatase
-
 Plasmodium falciparum
PfPPase
-
 Plasmodium falciparum
PPase
-
 Toxoplasma gondii
PPase
-
 Plasmodium falciparum
TgPPase
-
 Toxoplasma gondii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Toxoplasma gondii
37
-
 assay at Plasmodium falciparum

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.53
-
 diphosphate pH 7.2, 37°C, recombinant mutant D203N Plasmodium falciparum
21.8
-
 diphosphate pH 7.2, 37°C, recombinant mutant K136R Plasmodium falciparum
25.8
-
 diphosphate pH 7.2, 37°C, recombinant mutant D198N Plasmodium falciparum
52.5
-
 diphosphate pH 7.2, 37°C, recombinant mutant D235N Plasmodium falciparum
60.4
-
 diphosphate pH 7.2, 37°C, recombinant mutant R158K Plasmodium falciparum
266
-
 diphosphate pH 7.2, 37°C, recombinant wild-type enzyme Plasmodium falciparum

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7 7.2 optimal pHs for PPi, polyP3 and ATP hydrolysis are pH 7.2, pH 7.0, and pH 7.2 respectively Plasmodium falciparum
7.2
-
 assay at Toxoplasma gondii

General Information

General Information Comment Organism
evolution PPases include membrane associated V-H+-PPases (vacuolar H+-translocating PPases) and soluble form PPases, where latter comprise two families that differ in their sequence and structure. Family I PPases are Mg2+ dependent enzymes known to exist as homo-hexamers in prokaryotes and dimers in eukaryotes6. Family II PPases are Mn2+-dependent enzymes with bi-domain structures, and active in dimeric or trimeric forms. Structure comparisons of the enzyme from Plasmodium falciparum (PfPPase) and Toxoplasma gondii (TgPPase), overview. Comparison of eukaryotic family I PPases reveal diversity in dimerization modes Toxoplasma gondii
evolution PPases include membrane associated V-H+-PPases (vacuolar H+-translocating PPases) and soluble form PPases, where latter comprise two families that differ in their sequence and structure6. Family I PPases are Mg2+ dependent enzymes known to exist as homo-hexamers in prokaryotes and dimers in eukaryotes. Family II PPases are Mn2+ dependent enzymes with bi-domain structures, and active in dimeric or trimeric forms. Structure comparisons of the enzyme from Plasmodium falciparum (PfPPase) and Toxoplasma gondii (TgPPase), overview. Comparison of eukaryotic family I PPases reveals diversity in dimerization modes Plasmodium falciparum
malfunction deletion of the low complexity asparagine-rich N-terminal region has an unexpected and substantial effect on the stability of PfPPase domain, resulting in aggregation and significant loss of enzyme activity Plasmodium falciparum
additional information active site structure analysis Toxoplasma gondii
additional information active site structure analysis Plasmodium falciparum
physiological function the enzyme inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate (PPi) to inorganic phosphate (Pi). This is an exergonic reaction and can be coupled to several unfavorable and energy demanding biochemical transformations such as DNA replication, protein synthesis and lipid metabolism Toxoplasma gondii
physiological function the enzyme inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate (PPi) to inorganic phosphate (Pi). This is an exergonic reaction and can be coupled to several unfavorable and energy demanding biochemical transformations such as DNA replication, protein synthesis and lipid metabolism. Cambialistic PfPPase actively hydrolyzes linear short chain polyphosphates like diphosphate, polyP3, and ATP in the presence of Zn2+ Plasmodium falciparum

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
9.85
-
 diphosphate pH 7.2, 37°C, recombinant mutant D203N Plasmodium falciparum
54.12
-
 diphosphate pH 7.2, 37°C, recombinant mutant K136R Plasmodium falciparum
605.6
-
 diphosphate pH 7.2, 37°C, recombinant mutant D198N Plasmodium falciparum
940.8
-
 diphosphate pH 7.2, 37°C, recombinant mutant R158K Plasmodium falciparum
1148.8
-
 diphosphate pH 7.2, 37°C, recombinant mutant D235N Plasmodium falciparum
26600
-
 diphosphate pH 7.2, 37°C, recombinant wild-type enzyme Plasmodium falciparum