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Literature summary extracted from

  • Grzechowiak, M.; Ruszkowski, M.; Sliwiak, J.; Szpotkowski, K.; Sikorski, M.; Jaskolski, M.
    Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity (2019), Biochem. J., 476, 2297-2319 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.6.1.1 recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli Arabidopsis thaliana
3.6.1.1 recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli Medicago truncatula

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.6.1.1 purified recombinant enzyme, hanging drop vapor diffusion method, mixing of 7.5-10 mg/ml protein with precipitant solution containing 100 mM succinic acid, pH 7.0, and 15% PEG3350, or 100 mM bicine, pH 9.0, 7% PEG 6000, and 3 mM MgCl2 for the Mg2+-bound enzyme, 2 weeks, 19°C, 20% glycerol or PEG400 as cryoprotectants, X-ray diffraction structure determination and analysis at 1.93 A and 1.83 A resolution, respectively, structure modelling. Growth of AtPPA1 crystal is strongly correlated with the progression of proteolysis Arabidopsis thaliana
3.6.1.1 purified recombinant enzyme, hanging drop vapor diffusion method, mixing of 7.7 mg/ml bromelain-treated protein with precipitant solution containing 1.6% Tacsimate, pH 5.0, 80 mM tribasic sodium citrate, pH 5.6, 12.8% PEG 3350, and 20% glycerol, 2 days, 19°C, X-ray diffraction structure determination and analysis at 1.84-2.89A resolution, structure modelling Medicago truncatula

Protein Variants

EC Number Protein Variants Comment Organism
3.6.1.1 D103N site-directed mutagenesis Arabidopsis thaliana
3.6.1.1 D103N site-directed mutagenesis Medicago truncatula
3.6.1.1 D135N site-directed mutagenesis Arabidopsis thaliana
3.6.1.1 D135N site-directed mutagenesis Medicago truncatula
3.6.1.1 D98N site-directed mutagenesis Arabidopsis thaliana
3.6.1.1 D98N site-directed mutagenesis Medicago truncatula
3.6.1.1 additional information construction of an N-truncated variant of AtPPA1 with residues 1-29 deleted (DELTA(1-29)) produced using construct pMCSG48-AtPPA1-DELTA(1-29) Arabidopsis thaliana
3.6.1.1 additional information construction of the truncated version of MtPPA1-DELTA(1-30), which is not possible to express Medicago truncatula

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.6.1.1 0.0106
-
 diphosphate pH 7.5, 22°C, recombinant wild-type enzyme Medicago truncatula
3.6.1.1 0.0358
-
 diphosphate pH 7.5, 22°C, recombinant wild-type enzyme Arabidopsis thaliana
3.6.1.1 0.0432
-
 diphosphate pH 7.5, 22°C, recombinant truncated mutant AtPPA1-DELTA(1-29) Arabidopsis thaliana

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
3.6.1.1 mitochondrion the N-terminal peptide of immature AtPPA1 is mostly disordered. It can be cleaved during maturation. The cleaved peptide is a mitochondrial targeting signal Arabidopsis thaliana 5739
-
3.6.1.1 mitochondrion the N-terminal peptide of immature MtPPA1 can be cleaved during maturation. The cleaved peptide is a mitochondrial targeting signal Medicago truncatula 5739
-
3.6.1.1 soluble
-
 Arabidopsis thaliana
-
-
3.6.1.1 soluble
-
 Medicago truncatula
-
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.6.1.1 Mg2+ required, metal coordination and binding site Medicago truncatula
3.6.1.1 Mg2+ required, natural cofactor of AtPPA1, metal coordination and binding site Arabidopsis thaliana

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.6.1.1 132000
-
 gel filtration Arabidopsis thaliana
3.6.1.1 138000
-
 gel filtration Medicago truncatula

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.6.1.1 diphosphate + H2O Arabidopsis thaliana
-
 2 phosphate
-
 ?
3.6.1.1 diphosphate + H2O Medicago truncatula
-
 2 phosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.6.1.1 Arabidopsis thaliana Q93V56
-
-
3.6.1.1 Medicago truncatula I3STR7
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
3.6.1.1 proteolytic modification the N-terminal peptide is autocatalytically cleaved Medicago truncatula
3.6.1.1 proteolytic modification the N-terminal peptide is autocatalytically cleaved, presence of three N-terminal variants, truncated at Leu23, Ser25 and Leu26. The growth of AtPPA1 crystal iis strongly correlated with the progression of proteolysis Arabidopsis thaliana

Purification (Commentary)

EC Number Purification (Comment) Organism
3.6.1.1 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography, dialysis, tag cleavage by TEV protease, followed by gel filtration and ultrafiltration Arabidopsis thaliana
3.6.1.1 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography, dialysis, tag cleavage by TEV protease, followed by gel filtration and ultrafiltration Medicago truncatula

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.6.1.1 diphosphate + H2O
-
 Arabidopsis thaliana 2 phosphate
-
 ?
3.6.1.1 diphosphate + H2O
-
 Medicago truncatula 2 phosphate
-
 ?

Subunits

EC Number Subunits Comment Organism
3.6.1.1 homohexamer 6 * 24500, dimer of trimers, recombinant detagged enzyme, SDS-PAGE Medicago truncatula
3.6.1.1 homohexamer 6 * 25000, crecombinant detagged enzyme, SDS-PAGE Arabidopsis thaliana
3.6.1.1 More topology and conformation of the PPA1 subunits, comparison to the enzyme from Arabidospis thaliana Medicago truncatula
3.6.1.1 More topology and conformation of the PPA1 subunits, comparison to the enzyme from Medicago truncatula. The N-terminal peptide of immature AtPPA1 is mostly disordered Arabidopsis thaliana

Synonyms

EC Number Synonyms Comment Organism
3.6.1.1 AtPPA1
-
 Arabidopsis thaliana
3.6.1.1 inorganic pyrophosphatase
-
 Arabidopsis thaliana
3.6.1.1 inorganic pyrophosphatase
-
 Medicago truncatula
3.6.1.1 MtPPA1
-
 Medicago truncatula
3.6.1.1 PPase
-
 Arabidopsis thaliana
3.6.1.1 PPase
-
 Medicago truncatula

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.6.1.1 22
-
 assay at room temperature Arabidopsis thaliana
3.6.1.1 22
-
 assay at room temperature Medicago truncatula

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.6.1.1 5.3
-
 diphosphate pH 7.5, 22°C, recombinant wild-type enzyme Arabidopsis thaliana
3.6.1.1 10.8
-
 diphosphate pH 7.5, 22°C, recombinant wild-type enzyme Medicago truncatula
3.6.1.1 21.4
-
 diphosphate pH 7.5, 22°C, recombinant truncated mutant AtPPA1-DELTA(1-29) Arabidopsis thaliana

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.6.1.1 7.5
-
 assay at Arabidopsis thaliana
3.6.1.1 7.5
-
 assay at Medicago truncatula

General Information

EC Number General Information Comment Organism
3.6.1.1 additional information AtPPA1 three-dimensional structure analysis and modelling, overview Arabidopsis thaliana
3.6.1.1 additional information MtPPA1 three-dimensional structure analysis and modelling, overview Medicago truncatula
3.6.1.1 physiological function inorganic diphosphatases (PPases), which hydrolyze inorganic diphosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells Arabidopsis thaliana
3.6.1.1 physiological function inorganic diphosphatases (PPases), which hydrolyze inorganic diphosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells Medicago truncatula

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.6.1.1 148.1
-
 diphosphate pH 7.5, 22°C, recombinant wild-type enzyme Arabidopsis thaliana
3.6.1.1 495.4
-
 diphosphate pH 7.5, 22°C, recombinant truncated mutant AtPPA1-DELTA(1-29) Arabidopsis thaliana
3.6.1.1 1019
-
 diphosphate pH 7.5, 22°C, recombinant wild-type enzyme Medicago truncatula