Cloned (Comment) | Organism |
---|---|
gene siw14, recombinant expression of His6-MBP-tagged wild-type and mutant enzymes in Escherichia coli | Saccharomyces cerevisiae |
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 | 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 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 | UniProt | Comment | Textmining |
---|---|---|---|
Saccharomyces cerevisiae | - |
- |
- |
Saccharomyces cerevisiae ATCC 204508 | - |
- |
- |
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 | 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 | 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 Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Saccharomyces cerevisiae |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.2 | - |
assay at | Saccharomyces cerevisiae |
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 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 |