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Literature summary for 3.7.1.4 extracted from

  • Schoefer, L.; Braune, A.; Blaut, M.
    Cloning and Expression of a Phloretin Hydrolase Gene from Eubacterium ramulus and Characterization of the Recombinant Enzyme (2004), Appl. Environ. Microbiol., 70, 6131-6137.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
DNA and amino acid sequence determination and analysis, expression in Escherichia coli DH5alpha Eubacterium ramulus
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

Inhibitors Comment Organism Structure
1-bromopyrrolidine-2,5-dione 3% inhibition at 1 mM Eubacterium ramulus
3-(4-hydroxyphenyl)propionic acid i.e. phloretate, 30% product inhibition at 0.12 mM; product inhibition Eubacterium ramulus
Cu2+ 15% inhibition at 1 mM Eubacterium ramulus
CuCl2 85% inhibition at 1 mM Eubacterium ramulus
N-bromosuccinimide 97% inhibition at 1 mM Eubacterium ramulus
N-ethylmaleimide 65% inhibition at 1 mM Eubacterium ramulus
NEM 35% inhibition at 1 mM Eubacterium ramulus
o-phenanthroline 20% inhibition at 1 mM Eubacterium ramulus
phloroglucinol 46% product inhibition at 0.12 mM; product inhibition Eubacterium ramulus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.013
-
phloretin pH 7.0, 37°C Eubacterium ramulus
0.013
-
phloretin recombinant enzyme, pH 7.0, 37°C Eubacterium ramulus

Metals/Ions

Metals/Ions Comment Organism Structure
additional information no affected by Mg2+ and Ca2+ Eubacterium ramulus
additional information possibly a metal-dependent enzyme, no effects by Ca2+ or Mg2+ on the enzyme activity Eubacterium ramulus

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
30000
-
2 * 30846, sequence calculation, 2 * 30000, recombinant enzyme, SDS-PAGE Eubacterium ramulus
30846
-
2 * 30846, sequence calculation, 2 * 30000, recombinant enzyme, SDS-PAGE Eubacterium ramulus
55000
-
gel filtration Eubacterium ramulus
55000
-
recombinant enzyme, gel filtration Eubacterium ramulus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
phloretin + H2O Eubacterium ramulus
-
phloretate + phloroglucinol
-
?
phloretin + H2O Eubacterium ramulus during flavonoid degradation phloroglucinol + 3-(4-hydroxyphenyl)propionic acid
-
?
phloretin + H2O Eubacterium ramulus wK1
-
phloretate + phloroglucinol
-
?

Organism

Organism UniProt Comment Textmining
Eubacterium ramulus Q715L4
-
-
Eubacterium ramulus Q715L4 gene phy
-
Eubacterium ramulus wK1 Q715L4
-
-
Eubacterium ramulus wK1 Q715L4 gene phy
-

Purification (Commentary)

Purification (Comment) Organism
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
recombinant enzyme from Escherichia coli, about 13.2fold Eubacterium ramulus

Reaction

Reaction Comment Organism Reaction ID
phloretin + H2O = phloretate + phloroglucinol also hydrolyses other C-acetylated phenols related to phloretin Eubacterium ramulus

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.0064
-
cell extract Eubacterium ramulus
8.7
-
purified enzyme Eubacterium ramulus
8.7
-
purified recombinant enzyme, pH 7.0, 37°C Eubacterium ramulus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
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 ?
-
?
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 ?
-
?
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 ?
-
?
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 ?
-
?
phloretin + H2O
-
Eubacterium ramulus phloretate + phloroglucinol
-
?
phloretin + H2O a C-C bond cleaving enzyme Eubacterium ramulus phloretate + phloroglucinol
-
?
phloretin + H2O
-
Eubacterium ramulus wK1 phloretate + phloroglucinol
-
?
phloretin + H2O a C-C bond cleaving enzyme Eubacterium ramulus wK1 phloretate + phloroglucinol
-
?
phloretin + H2O
-
Eubacterium ramulus phloroglucinol + 3-(4-hydroxyphenyl)propionic acid
-
ir
phloretin + H2O during flavonoid degradation Eubacterium ramulus phloroglucinol + 3-(4-hydroxyphenyl)propionic acid
-
?
phloretin + H2O
-
Eubacterium ramulus wK1 phloroglucinol + 3-(4-hydroxyphenyl)propionic acid
-
ir

Subunits

Subunits Comment Organism
dimer 2 * 30000-31000, SDS-PAGE Eubacterium ramulus
homodimer 2 * 30846, sequence calculation, 2 * 30000, recombinant enzyme, SDS-PAGE Eubacterium ramulus

Synonyms

Synonyms Comment Organism
phloretin hydrolase
-
Eubacterium ramulus
Phy
-
Eubacterium ramulus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
-
Eubacterium ramulus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
10
-
phloretin pH 7.0, 37°C Eubacterium ramulus
10
-
phloretin recombinant enzyme, pH 7.0, 37°C Eubacterium ramulus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
-
Eubacterium ramulus

General Information

General Information Comment Organism
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
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