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Literature summary for 2.4.1.67 extracted from
- Molecular cloning of AtRS4, a seed specific multifunctional RFO synthase/galactosylhydrolase in Arabidopsis thaliana (2015), Front. Plant Sci., 6, 789.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expressed in Escherichia coli | Arabidopsis thaliana |
| gene RFS4, cloning from seeds and functional recombinant expression of His-tagged enzyme in Escherichia coli | Arabidopsis thaliana |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| additional information | isolation of a T-DNA insertional mutant in the AtRS4 gene, only semi-quantitative PCR from wild-type siliques shows a specific transcriptional AtRS4PCR product. Metabolite measurements in seeds of DELTAAtRS4 mutant plants reveal a total loss of stachyose in DELTAAtRS4 mutant seeds | Arabidopsis thaliana |
General Stability
| General Stability | Organism |
|---|---|
| Mg2+, bovine serum albumin, Triton, Tween 20, and DTT stabilize the enzyme, the latter is used in the enzyme assay | Arabidopsis thaliana |
| utilization of Na-phosphate and MES buffer (pH 7.0) leads to 86.5% and 82.5% of enzyme activity, respectively, compared with 100% enzyme activity in K-phosphate buffer at the same pH | Arabidopsis thaliana |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | Michaelis-Menten kinetics | Arabidopsis thaliana | |
| 0.2592 | - |
raffinose | at pH 7.0 and 25°C | Arabidopsis thaliana |  |
| 0.2592 | - |
raffinose | pH 7.0, 25°C, recombinant His-tagged enzyme | Arabidopsis thaliana | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | stabilizes the enzyme | Arabidopsis thaliana | |
| additional information | K-phosphate buffer is preferred | Arabidopsis thaliana |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 98001 | - |
1 * 98001, calculated from amino acid sequence | Arabidopsis thaliana |
| 98010 | - |
x * 98010, about, sequence calculation, x * 100000, recombinant His-tagged enzyme, SDS-PAGE | Arabidopsis thaliana |
| 100000 | - |
gel filtration | Arabidopsis thaliana |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| alpha-D-galactosyl-(1->3)-1D-myo-inositol + raffinose | Arabidopsis thaliana | - |
myo-inositol + stachyose | - |
? | |
| alpha-D-galactosyl-(1->3)-1D-myo-inositol + raffinose | Arabidopsis thaliana Col-0 | - |
myo-inositol + stachyose | - |
? | |
| additional information | Arabidopsis thaliana | the multifunctional enzyme is a raffinose and high affinity stachyose synthase, EC 2.4.1.82 and EC 2.4.1.67, as well as stachyose and galactinol specific galactosylhydrolase | ? | - |
? | |
| additional information | Arabidopsis thaliana Col-0 | the multifunctional enzyme is a raffinose and high affinity stachyose synthase, EC 2.4.1.82 and EC 2.4.1.67, as well as stachyose and galactinol specific galactosylhydrolase | ? | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Arabidopsis thaliana | - |
ecotype Columbia | - |
| Arabidopsis thaliana | - |
gene RFS4, isozyme RS4 | - |
| Arabidopsis thaliana Col-0 | - |
gene RFS4, isozyme RS4 | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| nickel affinity column chromatography | Arabidopsis thaliana |
| recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography | Arabidopsis thaliana |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|
| seed | - |
Arabidopsis thaliana | - |
| seed | isozyme RS4 is seed-specific | Arabidopsis thaliana | - |
| silique | - |
Arabidopsis thaliana | - |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 0.28 | - |
purified recombinant enzyme, pH 7.0, 25°C | Arabidopsis thaliana |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| alpha-D-galactosyl-(1->3)-1D-myo-inositol + raffinose | - |
Arabidopsis thaliana | myo-inositol + stachyose | - |
? | |
| alpha-D-galactosyl-(1->3)-1D-myo-inositol + raffinose | - |
Arabidopsis thaliana Col-0 | myo-inositol + stachyose | - |
? | |
| additional information | the multifunctional enzyme is a raffinose and high affinity stachyose synthase, EC 2.4.1.82 and EC 2.4.1.67, as well as stachyose and galactinol specific galactosylhydrolase | Arabidopsis thaliana | ? | - |
? | |
| additional information | the multifunctional enzyme is a raffinose and high affinity stachyose synthase, EC 2.4.1.82 and EC 2.4.1.67, as well as stachyose and galactinol specific galactosylhydrolase | Arabidopsis thaliana Col-0 | ? | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| ? | x * 98010, about, sequence calculation, x * 100000, recombinant His-tagged enzyme, SDS-PAGE | Arabidopsis thaliana |
| monomer | 1 * 98001, calculated from amino acid sequence | Arabidopsis thaliana |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| More | cf. EC 2.4.1.82 | Arabidopsis thaliana |
| RFO synthase/galactosylhydrolase | - |
Arabidopsis thaliana |
| RS4 | multifunctional enzyme | Arabidopsis thaliana |
| stachyose synthase | - |
Arabidopsis thaliana |
| StaS | - |
Arabidopsis thaliana |
| STS | - |
Arabidopsis thaliana |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | - |
- |
Arabidopsis thaliana |
| 25 | - |
measured at pH 8.0 | Arabidopsis thaliana |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 5 | 35 | activity range, inactive at 0°C and 40°C | Arabidopsis thaliana |
| 5 | 35 | at 5°C, 22.2% of enzyme activity is sustained, whereas 98.3% of the activity is lost at 40°C caused by protein inactivation | Arabidopsis thaliana |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 6 | - |
- |
Arabidopsis thaliana |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 6 | 8 | tested range, maximal activity at pH 6.0, 65.8% of maximal activity at pH 8.0, 89.5% at pH 7.0 | Arabidopsis thaliana |
| 7 | 8 | 89.5% and 65.8% of activity at pH 7.0 and pH 8.0, respectively | Arabidopsis thaliana |
General Information
| General Information | Comment | Organism |
|---|---|---|
| malfunction | metabolite phenotype of DELTAAtRS4 mutant seeds, overview | Arabidopsis thaliana |
| metabolism | biosynthesis of RFOs proceeds by stepwise transfer of galactosyl units. The second step involves raffinose synthase, AtRS5, EC 2.4.1.82, which transfers the galactosyl unit from galactinol to the C6 position of the glucose unit in sucrose forming an alpha-1,6-galactosidic linkage to yield the trisaccharide raffinose. In a third step, stachyose synthase, AtRS4, EC 2.4.1.67, transfers the galactosyl moiety from galactinol to the C6 position of the galactose unit in raffinose to yield the tetrasaccharide stachyose | Arabidopsis thaliana |
| physiological function | AtRS4 is the only stachyose synthase in the genome of Arabidopsis thaliana. Stachyose synthase is an important enzyme involved in the biosynthesis of raffinose family oligosaccharides, which occur frequently in higher plants. The enzyme represents a key regulation mechanism in the raffinose family oligosaccharide physiology of Arabidosis thaliana due to its multifunctional enzyme activity. AtRS4 is possibly the second seed-specific raffinose synthase beside AtRS5, which is responsible for raffinose accumulation under abiotic stress | Arabidopsis thaliana |
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