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Literature summary for 3.1.3.56 extracted from

  • Wang, H.; Gu, C.; Rolfes, R.J.; Jessen, H.J.; Shears, S.B.
    Structural and biochemical characterization of Siw14 a protein-tyrosine phosphatase fold that metabolizes inositol pyrophosphates (2018), J. Biol. Chem., 293, 6905-6914 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene siw14, recombinant expression of His6-MBP-tagged wild-type and mutant enzymes in Escherichia coli Saccharomyces cerevisiae

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant catalytic core of Siw14 (residues 116-281), hanging drop vapor diffusion, mixing of 0.002 ml of 15 mg/ml protein in 150 mM NaCl, and 20 mM Tris-HCl, pH 7.5, with 0.002 ml of well solution containing 0.7 M sodium citrate, and 50 mM 2-mercaptoethanol, 25°C, X-ray diffraction structure determination and analysis at 2.35-2.38 A resolution, model building Saccharomyces cerevisiae

Protein Variants

Protein Variants Comment Organism
C214S site-directed mutagenesis, mutation in the P-loop, the mutant shows altered substrate specificities and activities compared to the wild-type enzyme, overview Saccharomyces cerevisiae
E185A site-directed mutagenesis, mutation in the Flex-loop, the mutant shows altered substrate specificities and activities compared to the wild-type enzyme, overview Saccharomyces cerevisiae
F246A site-directed mutagenesis, mutation in the bulky and hydrophobic loop, inactive mutant Saccharomyces cerevisiae
H219A site-directed mutagenesis, mutation in the P-loop/alpha-helix4, the mutant shows altered substrate specificities and activities compared to the wild-type enzyme, overview Saccharomyces cerevisiae
K184A site-directed mutagenesis, mutation in the Flex-loop, the mutant shows altered substrate specificities and activities compared to the wild-type enzyme, overview Saccharomyces cerevisiae
K218A site-directed mutagenesis, mutation in the P-loop, the mutant shows altered substrate specificities and activities compared to the wild-type enzyme, overview Saccharomyces cerevisiae
K250A site-directed mutagenesis, mutation in the alpha4-alpha5-loop, the mutant shows altered substrate specificities and activities compared to the wild-type enzyme, overview Saccharomyces cerevisiae
N183A site-directed mutagenesis, mutation in the Flex-loop, the mutant shows altered substrate specificities and activities compared to the wild-type enzyme, overview Saccharomyces cerevisiae
N215A site-directed mutagenesis, mutation in the P-loop, the mutant shows altered substrate specificities and activities compared to the wild-type enzyme, overview Saccharomyces cerevisiae
R216A site-directed mutagenesis, mutation in the P-loop, the mutant shows altered substrate specificities and activities compared to the wild-type enzyme, overview Saccharomyces cerevisiae
R220A site-directed mutagenesis, mutation in the P-loop/alpha4-helix, the mutant shows altered substrate specificities and activities compared to the wild-type enzyme, overview Saccharomyces cerevisiae
R252A site-directed mutagenesis, mutation in the alpha4-alpha5-loop, the mutant shows altered substrate specificities and activities compared to the wild-type enzyme, overview Saccharomyces cerevisiae
W234A site-directed mutagenesis, mutation in the bulky and hydrophobic loop, the mutant is only active with 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate Saccharomyces cerevisiae
W281A site-directed mutagenesis, mutation in the bulky and hydrophobic loop, inactive mutant Saccharomyces cerevisiae

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.007
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant N215A Saccharomyces cerevisiae
0.0101
-
1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate pH 7.2, 30°C, wild-type enzyme Siw14 Saccharomyces cerevisiae
0.0102
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant N183A Saccharomyces cerevisiae
0.0104
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, wild-type enzyme Siw14 Saccharomyces cerevisiae
0.0116
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, recombinant truncated enzyme Siw14(116-281) Saccharomyces cerevisiae
0.0122
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant E185A Saccharomyces cerevisiae
0.0145
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant R216A Saccharomyces cerevisiae
0.017
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant H219A Saccharomyces cerevisiae
0.0286
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant K218A Saccharomyces cerevisiae
0.0326
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant K250A Saccharomyces cerevisiae
0.0471
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant K184A Saccharomyces cerevisiae
0.0481
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant R252A Saccharomyces cerevisiae
0.0568
-
5-diphosphoinositol 1,3,4,6-tetrakisphosphate pH 7.2, 30°C, recombinant wild-type enzyme Siw14 Saccharomyces cerevisiae

Organism

Organism UniProt Comment Textmining
Saccharomyces cerevisiae
-
-
-
Saccharomyces cerevisiae ATCC 204508
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-MBP-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity and heparin affinity chromatography, tag cleavage with TEV protease, and gel filtration Saccharomyces cerevisiae

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate + H2O moderate activity Saccharomyces cerevisiae 1-diphosphoinositol 2,3,4,6-tetrakisphosphate + phosphate
-
?
1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate + H2O moderate activity Saccharomyces cerevisiae ATCC 204508 1-diphosphoinositol 2,3,4,6-tetrakisphosphate + phosphate
-
?
1-diphosphoinositol 2,3,4,5,6-pentakisphosphate + H2O low activity Saccharomyces cerevisiae ?
-
?
1-diphosphoinositol 2,3,4,5,6-pentakisphosphate + H2O low activity Saccharomyces cerevisiae ATCC 204508 ?
-
?
4-diphosphoinositol 1,2,3,5,6-pentakisphosphate + H2O low activity Saccharomyces cerevisiae ?
-
?
4-diphosphoinositol 1,2,3,5,6-pentakisphosphate + H2O low activity Saccharomyces cerevisiae ATCC 204508 ?
-
?
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate + H2O best substrate Saccharomyces cerevisiae myo-inositol hexakisphosphate + phosphate
-
?
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate + H2O best substrate Saccharomyces cerevisiae ATCC 204508 myo-inositol hexakisphosphate + phosphate
-
?
5-diphosphoinositol 1,3,4,6-tetrakisphosphate + H2O moderate to low activity Saccharomyces cerevisiae inositol-1,3,4,5,6-pentakisphosphate + phosphate
-
?
6-diphosphoinositol 1,2,3,4,5-pentakisphosphate + H2O low activity Saccharomyces cerevisiae ?
-
?
additional information the enzyme shows high activity and exquisite specificity for the 5-diphosphate group of inositol diphosphates (5-PP-InsP), substrate-binding pocket structure analysis, overview. No activity with inositol hexakisphosphate, poor activity with 2-diphosphoinositol 1,3,4,5,6-pentakisphosphate and 3-diphosphoinositol 1,2,4,5,6-pentakisphosphate. Enzyme Siw14 also acts as protein tyrosine phosphatase, EC 3.1.3.48 Saccharomyces cerevisiae ?
-
?
additional information the enzyme shows high activity and exquisite specificity for the 5-diphosphate group of inositol diphosphates (5-PP-InsP), substrate-binding pocket structure analysis, overview. No activity with inositol hexakisphosphate, poor activity with 2-diphosphoinositol 1,3,4,5,6-pentakisphosphate and 3-diphosphoinositol 1,2,4,5,6-pentakisphosphate. Enzyme Siw14 also acts as protein tyrosine phosphatase, EC 3.1.3.48 Saccharomyces cerevisiae ATCC 204508 ?
-
?

Synonyms

Synonyms Comment Organism
DUSP
-
Saccharomyces cerevisiae
More cf. EC 3.1.3.48 Saccharomyces cerevisiae
protein-tyrosine phosphatase
-
Saccharomyces cerevisiae
PTP
-
Saccharomyces cerevisiae
Siw14
-
Saccharomyces cerevisiae
specific protein-tyrosine phosphatase
-
Saccharomyces cerevisiae
tyrosine-protein phosphatase
-
Saccharomyces cerevisiae

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Saccharomyces cerevisiae

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.21
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant K250A Saccharomyces cerevisiae
0.29
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant N215A Saccharomyces cerevisiae
0.35
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant R252A Saccharomyces cerevisiae
0.38
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant H219A Saccharomyces cerevisiae
0.46
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant K184A Saccharomyces cerevisiae
0.48
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant K218A Saccharomyces cerevisiae
0.57
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant N183A Saccharomyces cerevisiae
0.63
-
1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate pH 7.2, 30°C, wild-type enzyme Siw14 Saccharomyces cerevisiae
0.77
-
5-diphosphoinositol 1,3,4,6-tetrakisphosphate pH 7.2, 30°C, recombinant wild-type enzyme Siw14 Saccharomyces cerevisiae
0.8
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant E185A Saccharomyces cerevisiae
1.26
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant R216A Saccharomyces cerevisiae
1.31
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, wild-type enzyme Siw14 Saccharomyces cerevisiae
1.32
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, recombinant truncated enzyme Siw14(116-281) Saccharomyces cerevisiae

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.2
-
assay at Saccharomyces cerevisiae

General Information

General Information Comment Organism
evolution enzyme Siw14 is a member of the protein tyrosine-phosphatase (PTP) superfamily. Siw14 has a cysteine-based, class I CX5R(S/T) motif that defines the family of protein-tyrosine phosphatases (PTPs). Bioinformatic studies lead to Siw14 being classified as belonging within a specialist subgroup of PTPs, the dual specific protein-tyrosine phosphatases (DUSPs). The DUSPs themselves include a distinct class of proteins that appears not to have substantial activity against phosphoproteins. These are usually described as nonprotein-specific or atypical phosphatases. The inclusion of Siw14 in this category is supported by biochemical analysis, the enzyme's catalytic activity against 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) is several orders of magnitude greater than that against 4-nitrophenyl phosphate, a generic protein phosphatase substrate. Other members of this atypical DUSP subgroup preferentially hydrolyze either phosphorylated carbohydrates, inositol lipids, or triphosphate groups in mRNA. Thus, this DUSP subfamily exhibits catalytic site diversity that is not observed for classical PTPs Saccharomyces cerevisiae
metabolism inositol pyrophosphate metabolism in Saccharomyces cerevisiae involving enzyme Siw14, overview Saccharomyces cerevisiae
additional information active site architecture and substrate binding pocket structure, overview. The core catalytic domain of Siw14 is formed by residues 116-281. The three structural elements that demarcate a 9.2-A-deep substrate-binding pocket each have spatial equivalents in PTPs, but these are specialized for Siw14 to bind and hydrolyze the intensely negatively charged diphosphoinositol phosphates. A loop between the alpha5 and alpha6 helices, corresponding to the Q-loop in PTPs, contains a lysine and an arginine that extend into the catalytic pocket due to displacement of the alpha5 helix orientation through intramolecular crowding caused by three bulky, hydrophobic residues. The general-acid loop in PTPs is replaced in Siw14 with a flexible loop that does not use an aspartate or glutamate as a general acid Saccharomyces cerevisiae

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
6.44
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant K250A Saccharomyces cerevisiae
7.28
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant R252A Saccharomyces cerevisiae
9.77
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant K184A Saccharomyces cerevisiae
13.56
-
5-diphosphoinositol 1,3,4,6-tetrakisphosphate pH 7.2, 30°C, recombinant wild-type enzyme Siw14 Saccharomyces cerevisiae
16.78
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant K218A Saccharomyces cerevisiae
22.36
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant H219A Saccharomyces cerevisiae
41.43
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant N215A Saccharomyces cerevisiae
55.88
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant N183A Saccharomyces cerevisiae
62.38
-
1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate pH 7.2, 30°C, wild-type enzyme Siw14 Saccharomyces cerevisiae
65.57
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant E185A Saccharomyces cerevisiae
86.9
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, Siw14 mutant R216A Saccharomyces cerevisiae
113.79
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, recombinant truncated enzyme Siw14(116-281) Saccharomyces cerevisiae
125.96
-
5-diphosphoinositol 1,2,3,4,6-pentakisphosphate pH 7.2, 30°C, wild-type enzyme Siw14 Saccharomyces cerevisiae