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Literature summary for 2.4.1.170 extracted from

  • Noguchi, A.; Saito, A.; Homma, Y.; Nakao, M.; Sasaki, N.; Nishino, T.; Takahashi, S.; Nakayama, T.
    A UDP-glucose:isoflavone 7-O-glucosyltransferase from the roots of soybean (Glycine max) seedlings: Purification, gene cloning, phylogenetics, and an implication for an alternative strategy of enzyme catalysis (2007), J. Biol. Chem., 282, 23581-23590.
    View publication on PubMed
Search for more literature for 2.4.1.170

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) Glycine max

Protein Variants

Protein Variants Comment Organism
D125A in the mutant the kcat is lower and the Km is higher than in wild type enzyme Glycine max
E376A in the mutant the kcat is lower and the Km is higher than in wild type enzyme Glycine max
E392A in the mutant the kcat is lower and the Km is higher than in wild type enzyme Glycine max
E392D in the mutant the kcat is higher and the Km is lower than in wild type enzyme Glycine max
E456A in the mutant the kcat is lower and the Km is slightly lower than in wild type enzyme Glycine max
H15A in the mutant the kcat is lower and the Km is higher than in wild type enzyme Glycine max
H359A in the mutant the kcat is lower and the Km has the same level as in wild type enzyme Glycine max
H368A in the mutant the kcat is higher and the Km is higher than in wild type enzyme Glycine max

Inhibitors

Inhibitors Comment Organism Structure
Fe2+ 0.1 mM, residual activity 28% Glycine max
Hg2+ 0.1 mM, residual activity 26% Glycine max
additional information 1 mM EDTA, 1mM uridine, 1 mM UMP, 1 mM UDP, 1 mM UTP, 1 mM glucose, 1 mM diethylpyrocarbonatem or 1 mM phenylmethanesulfonyl fluoride have negligibel effect on the catalytic activity (residual activity, in excess of 64%). Glycine max

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.00077
-
 genistein E392D mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.0026
-
 genistein E456A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.0027
-
 genistein H359A muant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.0027
-
 genistein wild-type, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.0036
-
 genistein purified enzyme, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.0045
-
 genistein E376A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.0049
-
 genistein H368A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.011
-
 genistein E392A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.016
-
 genistein H15A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.024
-
 UDP-glucose E392D mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.027
-
 UDP-glucose H15A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.045
-
 UDP-glucose D125A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.06
-
 genistein D125A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.072
-
 UDP-glucose H359A muant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.081
-
 UDP-glucose H368A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.082
-
 UDP-glucose E456A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.13
-
 UDP-glucose wild-type, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.19
-
 UDP-glucose E392A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.19
-
 UDP-glucose purified enzyme, enzyme assay is run at pH 8.5 at 30°C Glycine max
2.1
-
 UDP-glucose E376A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max

Metals/Ions

Metals/Ions Comment Organism Structure
additional information Ca2+, Cd2+, Co2+, Cu2+, Mg2+, Mn2+, Ni2+, Sn2+ or Zn2+ (all at 0.1 mM) have negligible effect on the catalytic activity. Glycine max

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
45000
-
 SDS-PAGE Glycine max
46000
-
 gel filtration chromatography Glycine max

Organism

Organism UniProt Comment Textmining
Glycine max
-
 soybean, Merr. cv. Wase-Hakucho
-

Purification (Commentary)

Purification (Comment) Organism
DEAE-Sepharose Fast Flow column, Q-Sepharose HP column, Phenyl-Sepharose HP column (pH 8.5), hydroxyapatite column, gel filtration (HiLoad 26/60 Superdex 200pg column), resource Q column and chromatofocusing on Mono P (Mono P column), all at 4°C. The activity in the crude extract is extremely unstable to oxidation and the addition of 2-mercaptoethanol is essential for its efficient purification. The addition of CHAPS to purification buffers is also essential for purification after step 6 (hydroxyapatite chromatography), otherwise the activity is irreversibly lost. Glycine max

Source Tissue

Source Tissue Comment Organism Textmining
root roots of 7-9 day-old seedlings Glycine max
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 as glucosyl acceptor genistein shows the highest activity (100%), daidzein has 73%, formononetin shows 67%, quercetin 32%, kaempferol 19%, 4,2',4',6'-tetrahydoxychalcone 15%, apigenin 14%, aureusidin 5%, esculetin 4% and naringenin shows 3% activity (all tested with UDP-glucose). Enzyme assay is run at pH 8.5 at 30°C Glycine max

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information none of the following phenolics serve as glucosyl acceptors: cyanidin, trans-p-coumaric acid, caffeic acid, benzoic acid, m- and p-hydroybenzoic acid, salicylic acid, salicyl alcohol and hydroquinone. Glycine max ?
-
 ?
additional information the relative activites for UDP-glucuronic acid and for UDP-galactose is 0% and for UDP-glucose 12%, all tested with genistein as glycosyl acceptor Glycine max ?
-
 ?
UDP-glucose + 4,2',4',6'-tetrahydroxychalcone
-
 Glycine max UDP + ?
-
 ?
UDP-glucose + apigenin
-
 Glycine max UDP + apigenin 7-O-beta-D-glucoside
-
 ?
UDP-glucose + aureusidin
-
 Glycine max UDP + aureusidin 6-O-beta-D-glucoside
-
 ?
UDP-glucose + daidzein
-
 Glycine max UDP + daidzein 7-O-beta-D-glucoside
-
 ?
UDP-glucose + esculetin
-
 Glycine max UDP + esculetin 7-O-beta-D-glucoside
-
 ?
UDP-glucose + formononetin
-
 Glycine max UDP + formononetin 7-O-beta-D-glucoside
-
 ?
UDP-glucose + genistein
-
 Glycine max UDP + genistein 7-O-beta-D-glucoside
-
 ?
UDP-glucose + kaempferol
-
 Glycine max UDP + kaempferol 7-O-beta-D-glucoside
-
 ?
UDP-glucose + naringenin
-
 Glycine max UDP + naringenin 7-O-beta-D-glucoside
-
 ?
UDP-glucose + quercetin
-
 Glycine max UDP + quercetin 7-O-beta-D-glucoside
-
 ?

Subunits

Subunits Comment Organism
monomer
-
 Glycine max

Synonyms

Synonyms Comment Organism
GmIF7GT UDP-glucose:isoflavone 7-O-glucosyltransferase Glycine max

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
 below 30°C at pH 8.5 for 1h Glycine max

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0004
-
 genistein E392A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.13
-
 genistein H15A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.41
-
 genistein E456A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.59
-
 genistein H359A muant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.63
-
 genistein E376A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.66
-
 genistein D125A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.74
-
 genistein purified enzyme, enzyme assay is run at pH 8.5 at 30°C Glycine max
0.76
-
 genistein wild-type, enzyme assay is run at pH 8.5 at 30°C Glycine max
2
-
 genistein H368A mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max
3
-
 genistein E392D mutant, enzyme assay is run at pH 8.5 at 30°C Glycine max

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.5
-
 optimal pH at 30°C Glycine max

pH Range

pH Minimum pH Maximum Comment Organism
7 9 active over this pH range Glycine max

pH Stability

pH Stability pH Stability Maximum Comment Organism
5.5 10 at 20°C for 8 h Glycine max