Literature summary for 3.6.1.1 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 .

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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
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

Crystallization (Comment)

Organism

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

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

Protein Variants	Comment	Organism
D103N	site-directed mutagenesis	Arabidopsis thaliana
D103N	site-directed mutagenesis	Medicago truncatula
D135N	site-directed mutagenesis	Arabidopsis thaliana
D135N	site-directed mutagenesis	Medicago truncatula
D98N	site-directed mutagenesis	Arabidopsis thaliana
D98N	site-directed mutagenesis	Medicago truncatula
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
additional information	construction of the truncated version of MtPPA1-DELTA(1-30), which is not possible to express	Medicago truncatula

KM Value [mM]

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

Localization

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

Metals/Ions

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

Molecular Weight [Da]

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Arabidopsis thaliana	Q93V56	-	-
Medicago truncatula	I3STR7	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
proteolytic modification	the N-terminal peptide is autocatalytically cleaved	Medicago truncatula
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)

Purification (Comment)	Organism
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
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)

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

Subunits

Subunits	Comment	Organism
homohexamer	6 * 24500, dimer of trimers, recombinant detagged enzyme, SDS-PAGE	Medicago truncatula
homohexamer	6 * 25000, crecombinant detagged enzyme, SDS-PAGE	Arabidopsis thaliana
More	topology and conformation of the PPA1 subunits, comparison to the enzyme from Arabidospis thaliana	Medicago truncatula
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

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

General Information

General Information	Comment	Organism
additional information	AtPPA1 three-dimensional structure analysis and modelling, overview	Arabidopsis thaliana
additional information	MtPPA1 three-dimensional structure analysis and modelling, overview	Medicago truncatula
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
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]

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