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Literature summary extracted from
- Cloning and Expression of a Phloretin Hydrolase Gene from Eubacterium ramulus and Characterization of the Recombinant Enzyme (2004), Appl. Environ. Microbiol., 70, 6131-6137.
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 3.7.1.4 | DNA and amino acid sequence determination and analysis, expression in Escherichia coli DH5alpha | Eubacterium ramulus |
| 3.7.1.4 | gene phy, encoded in the 2,4-diacetylphloroglucinol operon, library screening, DNA and amino acid sequence determination and analysis, sequence comparisons and promoter sequence determination, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain DH5alpha(pPH3) | Eubacterium ramulus |
Inhibitors
| EC Number | Inhibitors | Comment | Organism | Structure |
|---|---|---|---|---|
| 3.7.1.4 | 1-bromopyrrolidine-2,5-dione | 3% inhibition at 1 mM | Eubacterium ramulus |  |
| 3.7.1.4 | 3-(4-hydroxyphenyl)propionic acid | i.e. phloretate, 30% product inhibition at 0.12 mM; product inhibition | Eubacterium ramulus | |
| 3.7.1.4 | Cu2+ | 15% inhibition at 1 mM | Eubacterium ramulus | |
| 3.7.1.4 | CuCl2 | 85% inhibition at 1 mM | Eubacterium ramulus | |
| 3.7.1.4 | N-bromosuccinimide | 97% inhibition at 1 mM | Eubacterium ramulus | |
| 3.7.1.4 | N-ethylmaleimide | 65% inhibition at 1 mM | Eubacterium ramulus | |
| 3.7.1.4 | NEM | 35% inhibition at 1 mM | Eubacterium ramulus | |
| 3.7.1.4 | o-phenanthroline | 20% inhibition at 1 mM | Eubacterium ramulus | |
| 3.7.1.4 | phloroglucinol | 46% product inhibition at 0.12 mM; product inhibition | Eubacterium ramulus | |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 3.7.1.4 | 0.013 | - |
phloretin | pH 7.0, 37°C | Eubacterium ramulus | |
| 3.7.1.4 | 0.013 | - |
phloretin | recombinant enzyme, pH 7.0, 37°C | Eubacterium ramulus | |
Metals/Ions
| EC Number | Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|---|
| 3.7.1.4 | additional information | no affected by Mg2+ and Ca2+ | Eubacterium ramulus | |
| 3.7.1.4 | additional information | possibly a metal-dependent enzyme, no effects by Ca2+ or Mg2+ on the enzyme activity | Eubacterium ramulus |
Molecular Weight [Da]
| EC Number | Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|---|
| 3.7.1.4 | 30000 | - |
2 * 30846, sequence calculation, 2 * 30000, recombinant enzyme, SDS-PAGE | Eubacterium ramulus |
| 3.7.1.4 | 30846 | - |
2 * 30846, sequence calculation, 2 * 30000, recombinant enzyme, SDS-PAGE | Eubacterium ramulus |
| 3.7.1.4 | 55000 | - |
gel filtration | Eubacterium ramulus |
| 3.7.1.4 | 55000 | - |
recombinant enzyme, gel filtration | Eubacterium ramulus |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 3.7.1.4 | phloretin + H2O | Eubacterium ramulus | - |
phloretate + phloroglucinol | - |
? | |
| 3.7.1.4 | phloretin + H2O | Eubacterium ramulus | during flavonoid degradation | phloroglucinol + 3-(4-hydroxyphenyl)propionic acid | - |
? | |
| 3.7.1.4 | phloretin + H2O | Eubacterium ramulus wK1 | - |
phloretate + phloroglucinol | - |
? | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 3.7.1.4 | Eubacterium ramulus | Q715L4 | - |
- |
| 3.7.1.4 | Eubacterium ramulus | Q715L4 | gene phy | - |
| 3.7.1.4 | Eubacterium ramulus wK1 | Q715L4 | - |
- |
| 3.7.1.4 | Eubacterium ramulus wK1 | Q715L4 | gene phy | - |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 3.7.1.4 | recombinant enzyme 13.2fold from Escherichia coli strain DH5alpha(pPH3) by ammonium sulfate fractionation, anion exchange and hydrophobic interaction chromatography, followed by gel filtration | Eubacterium ramulus |
| 3.7.1.4 | recombinant enzyme from Escherichia coli, about 13.2fold | Eubacterium ramulus |
Reaction
| EC Number | Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|---|
| 3.7.1.4 | phloretin + H2O = phloretate + phloroglucinol | also hydrolyses other C-acetylated phenols related to phloretin | Eubacterium ramulus |
Specific Activity [micromol/min/mg]
| EC Number | Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|---|
| 3.7.1.4 | 0.0064 | - |
cell extract | Eubacterium ramulus |
| 3.7.1.4 | 8.7 | - |
purified enzyme | Eubacterium ramulus |
| 3.7.1.4 | 8.7 | - |
purified recombinant enzyme, pH 7.0, 37°C | Eubacterium ramulus |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 3.7.1.4 | additional information | no activity with phloretin-2'-glucoside, i.e. phloridzin, neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, and trans-1,3-diphenyl-2,3-epoxy-propan-1-one | Eubacterium ramulus | ? | - |
? | |
| 3.7.1.4 | additional information | no activity with glycosides phloretin-2'-glucoside (phloridzin) and neohesperidin dihydrochalcone, nor with compounds showing a structure similar to phloretin, such as 1,3-diphenyl-1,3-propandione and trans-1,3-diphenyl-2,3-epoxy-propan-1-one | Eubacterium ramulus | ? | - |
? | |
| 3.7.1.4 | additional information | no activity with phloretin-2'-glucoside, i.e. phloridzin, neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, and trans-1,3-diphenyl-2,3-epoxy-propan-1-one | Eubacterium ramulus wK1 | ? | - |
? | |
| 3.7.1.4 | additional information | no activity with glycosides phloretin-2'-glucoside (phloridzin) and neohesperidin dihydrochalcone, nor with compounds showing a structure similar to phloretin, such as 1,3-diphenyl-1,3-propandione and trans-1,3-diphenyl-2,3-epoxy-propan-1-one | Eubacterium ramulus wK1 | ? | - |
? | |
| 3.7.1.4 | phloretin + H2O | - |
Eubacterium ramulus | phloretate + phloroglucinol | - |
? | |
| 3.7.1.4 | phloretin + H2O | a C-C bond cleaving enzyme | Eubacterium ramulus | phloretate + phloroglucinol | - |
? | |
| 3.7.1.4 | phloretin + H2O | - |
Eubacterium ramulus wK1 | phloretate + phloroglucinol | - |
? | |
| 3.7.1.4 | phloretin + H2O | a C-C bond cleaving enzyme | Eubacterium ramulus wK1 | phloretate + phloroglucinol | - |
? | |
| 3.7.1.4 | phloretin + H2O | - |
Eubacterium ramulus | phloroglucinol + 3-(4-hydroxyphenyl)propionic acid | - |
ir | |
| 3.7.1.4 | phloretin + H2O | during flavonoid degradation | Eubacterium ramulus | phloroglucinol + 3-(4-hydroxyphenyl)propionic acid | - |
? | |
| 3.7.1.4 | phloretin + H2O | - |
Eubacterium ramulus wK1 | phloroglucinol + 3-(4-hydroxyphenyl)propionic acid | - |
ir | |
Subunits
| EC Number | Subunits | Comment | Organism |
|---|---|---|---|
| 3.7.1.4 | dimer | 2 * 30000-31000, SDS-PAGE | Eubacterium ramulus |
| 3.7.1.4 | homodimer | 2 * 30846, sequence calculation, 2 * 30000, recombinant enzyme, SDS-PAGE | Eubacterium ramulus |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 3.7.1.4 | phloretin hydrolase | - |
Eubacterium ramulus |
| 3.7.1.4 | Phy | - |
Eubacterium ramulus |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 3.7.1.4 | 37 | - |
- |
Eubacterium ramulus |
Turnover Number [1/s]
| EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 3.7.1.4 | 10 | - |
phloretin | pH 7.0, 37°C | Eubacterium ramulus | |
| 3.7.1.4 | 10 | - |
phloretin | recombinant enzyme, pH 7.0, 37°C | Eubacterium ramulus | |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 3.7.1.4 | 7 | - |
- |
Eubacterium ramulus |
General Information
| EC Number | General Information | Comment | Organism |
|---|---|---|---|
| 3.7.1.4 | evolution | C-C-cleaving hydrolases catalyzing reactions similar to the reaction carried out by phloretin hydrolase belong to the alpha/beta-hydrolase fold superfamily, but since the phloretin hydrolase sequence of Eubacterium ramulus has no sequence similarity to other C-C-cleaving hydrolases and also does not contain the conserved sequence motif GXSXG, it can be assumed that this enzyme belongs to another enzyme family. The sequences of phloretin hydrolase, Phy, and 2,4-diacetylphloroglucinol hydrolase, PhlG, show no similarities to any other protein sequences with known functions deposited in database | Eubacterium ramulus |
| 3.7.1.4 | physiological function | phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl) propionic acid during flavonoid degradation in Eubacterium ramulus | Eubacterium ramulus |
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