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Literature summary for 3.2.1.21 extracted from

  • Verdoucq, L.; Czjzek, M.; Moriniere, J.; Bevan, D.R.; Esen, A.
    Mutational and structural analysis of aglycone specificity in maize and sorghum beta-glucosidases (2003), J. Biol. Chem., 278, 25055-25062.
    View publication on PubMed
Search for more literature for 3.2.1.21

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli as a His-tagged fusion protein Sorghum bicolor
expressed in Escherichia coli as a His-tagged fusion protein Zea mays

Crystallization (Commentary)

Crystallization (Comment) Organism
crystal structure of mutant E191D,F198V is determined at 1.9 A resolution in complex with DIMBOA-glucoside Zea mays

Protein Variants

Protein Variants Comment Organism
A375P Km (2-nitrophenyl beta-D-glucopyranoside) decreased compared to wild-type, kcat (2-nitrophenyl beta-D-glucopyranoside) decreased. Relative efficacy toward (2-nitrophenyl beta-D-glucopyranoside) similar to wild-type. Km (dhurrin) similar to wild-type, kcat drastically decreased Sorghum bicolor
D261N the D261N substitution leads to a drastic decrease in relative efficiency of ZmGlu1 on all substrates tested, but little effect on the Km Zea mays
F198V mutant shows drastic effect on catalytic efficacy, kcat is reduced by 90% for hydrolysis of 4-methylumbelliferyl beta-D-glucopyranoside compared to wild-type. Mutant is unable to hydrolyze 4-nitrophenyl beta-D-glucopyranoside. Only negligible activity toward substrate 2-nitrophenyl beta-D-glucopyranoside compared to wild-type Zea mays
F198V/F205L/P377A mutant shows a drastic reduced relative efficacy for all test substrated due to a decreased turnover Zea mays
F205L Mutant shows similar relative efficacy compared to wild-type toward all substrates tested. Mutant shows no significant effect on hydrolysis of 4-nitrophenyl beta-D-glucopyranoside other than doubling the kcat value compared to wild-type Zea mays
F466S mutant exhibits a marked increase in relative efficiency in comparison to wild-type ZmGlu1 on all substrates tested (from 28 to 123%), because of increased substrate turnover. Mutant has little effect on dhurrin hydrolysis compared to wild-type (3% of relative efficacy compared to Sorghum Dhr1) Zea mays
F466S/A467S the A467S substitution in ZmGlu1 has no detectable effect on substrate specificity and catalytic efficiency in the case of 4-nitrophenyl beta-D-glucopyranoside or 2-nitrophenyl beta-D-glucopyranoside hydrolysis, and only a lower Km is obtained for 4-methylumbelliferyl beta-D-glucuronide compared to mutant F466S. Mutant has little effect on dhurrin hydrolysis compared to wild-type (3% of relative efficacy compared to Sorghum Dhr1) Zea mays
F469Y Km (2-nitrophenyl beta-D-glucopyranoside) increased compared to wild-type, kcat (2-nitrophenyl beta-D-glucopyranoside) decreased. Relative efficacy toward (2-nitrophenyl beta-D-glucopyranoside) significantly decreased by factor 2.5. Km (dhurrin) similar to wild-type, kcat drastically decreased Sorghum bicolor
M263F mutant shows reduced relative efficacy by 40 to 50% on 4-nitrophenyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-glucopyranoside whereas it shows an increase of about 60% relative efficacy on 2-nitrophenyl beta-D-glucopyranoside compared to wild-type Zea mays
additional information creation of chimeric mutants (Chim21 and Chim16) which are produced by reciprocal domain-swapping between ZmGlu1 and (Sorghum) SbDhr1: ZmGlu1 hydrolyzes dhurrin with a lower catalytic efficiency only when a C-terminal octapeptide 466-477 is exchanged with the homologous octapeptide of SorghumDhr1 (462-469). This peptide substitution leading to amino acid substitutions at four sites is found in Chim21 and Chim16 (with an extra 22 amino acids substitution) enables ZmGlu1 to hydrolyze dhurrin with less than 10% of the relative efficiency of SorghumDhr1 (7.4% for Chim21 and 9.5% for Chim16) Zea mays
P377A P377A mutation leads to changes (decreases) in both kcat and Km for all substrated with no decrease in the overall relative efficiency Zea mays
V196F/L203F Km (2-nitrophenyl beta-D-glucopyranoside) decreased compared to wild-type, kcat (2-nitrophenyl beta-D-glucopyranoside) decreased. Relative efficacy toward (2-nitrophenyl beta-D-glucopyranoside) similar to wild-type. Km (dhurrin) similar to wild-type, kcat drastically decreased Sorghum bicolor
Y473F kcat is increased by 110% to 450% depending on the substrate tested. Enhanced turnover is compensated by a slight increase in Km of this mutant giving a 40% to 250% (depending on the substrate tested) better relative efficacy over the wild-type enzyme. Mutation leads to a significant effect towards dhurrin hydrolysis (10% relative efficacy as compared to Sorghum Dhr1) Zea mays

Inhibitors

Inhibitors Comment Organism Structure
Dhurrin competitive inhibitor Zea mays

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.031
-
 Dhurrin pH 5.8, 37°C, mutant F205L Zea mays
0.047
-
 Dhurrin pH 5.8, 37°C, mutant Y473F Zea mays
0.058
-
 Dhurrin pH 5.8, 37°C, mutant F198V/F205L/P377A Zea mays
0.06
-
 Dhurrin pH 5.8, 37°C, mutant A375P Sorghum bicolor
0.061
-
 Dhurrin pH 5.8, 37°C, wild-type Sorghum bicolor
0.065
-
 Dhurrin pH 5.8, 37°C, mutant F466S/A467S Zea mays
0.065
-
 Dhurrin pH 5.8, 37°C, mutant V196F/L203F Sorghum bicolor
0.066
-
 Dhurrin pH 5.8, 37°C, mutant M263F Zea mays
0.076
-
 Dhurrin pH 5.8, 37°C, chimeric mutant (Chim21) bearing a C-terminal octapeptide (466-477) exchanged with the homologous octapeptide of Sorghum Dhr1 Zea mays
0.092
-
 Dhurrin pH 5.8, 37°C, mutant F466S Zea mays
0.101
-
 Dhurrin pH 5.8, 37°C, chimeric mutant (Chim16) bearing a C-terminal octapeptide (466-477) exchanged with the homologous octapeptide of Sorghum Dhr1 (with an extra 22 amino acids substitution) Zea mays
0.127
-
 Dhurrin pH 5.8, 37°C, mutant F469Y Sorghum bicolor
0.16
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant P377A Zea mays
0.23
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F198V Zea mays
0.33
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F198V/F205L/P377A Zea mays
0.37
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, wild-type Zea mays
0.395
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F205L Zea mays
0.41
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant M263F Zea mays
0.42
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant D261N Zea mays
0.43
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant M263F Zea mays
0.45
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant P377A Zea mays
0.49
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F466S Zea mays
0.55
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant Y473F Zea mays
0.625
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F205L Zea mays
0.64
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, wild-type Zea mays
0.76
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F466S/A467S Zea mays
0.9
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant D261N Zea mays
0.94
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F198V/F205L/P377A Zea mays
1.04
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant Y473F Zea mays
1.06
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F466S Zea mays
1.15
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F466S/A467S Zea mays
1.21
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant M263F Zea mays
1.22
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F198V Zea mays
1.24
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant P377A Zea mays
1.65
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, wild-type Zea mays
1.7
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant D261N Zea mays
1.85
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant Y473F Zea mays
1.86
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F205L Zea mays
1.87
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F466S Zea mays
1.88
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F466S/A467S Zea mays
2.72
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant A375P Sorghum bicolor
2.81
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant V196F/L203F Sorghum bicolor
3.76
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F198V/F205L/P377A Zea mays
3.83
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, wild-type Sorghum bicolor
5.51
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F469Y Sorghum bicolor

Organism

Organism UniProt Comment Textmining
Sorghum bicolor
-
-
-
Zea mays P49235
-
-

Purification (Commentary)

Purification (Comment) Organism
using Ni-NTA chromatography Sorghum bicolor
using Ni-NTA chromatography Zea mays

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-nitrophenyl beta-D-glucopyranoside + H2O
-
 Sorghum bicolor 2-nitrophenol + beta-D-glucose
-
 ?
2-nitrophenyl beta-D-glucopyranoside + H2O
-
 Zea mays 2-nitrophenol + beta-D-glucose
-
 ?
4-methylumbelliferyl beta-D-glucopyranoside + H2O
-
 Zea mays 4-methylumbelliferone + beta-D-glucose
-
 ?
4-nitrophenyl beta-D-glucopyranoside + H2O
-
 Zea mays 4-nitrophenol + beta-D-glucose
-
 ?
dhurrin + H2O
-
 Sorghum bicolor (2S)-hydroxy(4-hydroxyphenyl)ethanenitrile + beta-D-glucose
-
 ?
dhurrin + H2O
-
 Zea mays (2S)-hydroxy(4-hydroxyphenyl)ethanenitrile + beta-D-glucose
-
 ?

Subunits

Subunits Comment Organism
homodimer crystal structure Zea mays

Synonyms

Synonyms Comment Organism
SbDhr1
-
 Sorghum bicolor
ZmGlu1
-
 Zea mays

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Sorghum bicolor
37
-
 assay at Zea mays

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.16
-
 Dhurrin pH 5.8, 37°C, mutant F205L Zea mays
0.21
-
 Dhurrin pH 5.8, 37°C, mutant M263F Zea mays
0.55
-
 Dhurrin pH 5.8, 37°C, mutant F466S/A467S Zea mays
0.59
-
 Dhurrin pH 5.8, 37°C, mutant F466S Zea mays
0.67
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F198V/F205L/P377A Zea mays
0.7
-
 Dhurrin pH 5.8, 37°C, mutant F198V/F205L/P377A Zea mays
1.13
-
 Dhurrin pH 5.8, 37°C, mutant Y473F Zea mays
1.32
-
 Dhurrin pH 5.8, 37°C, chimeric mutant (Chim21) bearing a C-terminal octapeptide (466-477) exchanged with the homologous octapeptide of Sorghum Dhr1 Zea mays
1.7
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F198V/F205L/P377A Zea mays
2.23
-
 Dhurrin pH 5.8, 37°C, chimeric mutant (Chim16) bearing a C-terminal octapeptide (466-477) exchanged with the homologous octapeptide of Sorghum Dhr1 (with an extra 22 amino acids substitution) Zea mays
3.4
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F198V Zea mays
3.6
-
 Dhurrin pH 5.8, 37°C, mutant A375P Sorghum bicolor
3.7
-
 Dhurrin pH 5.8, 37°C, mutant V196F/L203F Sorghum bicolor
4.97
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant D261N Zea mays
6.52
-
 Dhurrin pH 5.8, 37°C, mutant F469Y Sorghum bicolor
6.6
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F198V Zea mays
9.2
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F469Y Sorghum bicolor
10.1
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant V196F/L203F Sorghum bicolor
10.85
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant P377A Zea mays
11.1
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant M263F Zea mays
11.4
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant A375P Sorghum bicolor
13.8
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, wild-type Sorghum bicolor
14.3
-
 Dhurrin pH 5.8, 37°C, wild-type Sorghum bicolor
16.8
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant P377A Zea mays
17.2
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant D261N Zea mays
17.8
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant M263F Zea mays
19.1
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F198V/F205L/P377A Zea mays
29.5
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, wild-type Zea mays
30.3
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, wild-type Zea mays
34.3
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F205L Zea mays
36.4
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant D261N Zea mays
57.3
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F205L Zea mays
63.4
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant Y473F Zea mays
74.4
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant P377A Zea mays
87.2
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F466S/A467S Zea mays
97.9
-
 4-methylumbelliferyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F466S Zea mays
100.7
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F466S Zea mays
118.5
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F466S/A467S Zea mays
121.5
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, wild-type Zea mays
137.3
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F205L Zea mays
143.8
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant M263F Zea mays
163.8
-
 4-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant Y473F Zea mays
172.4
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F466S Zea mays
177.1
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant F466S/A467S Zea mays
353.2
-
 2-nitrophenyl beta-D-glucopyranoside pH 5.8, 37°C, mutant Y473F Zea mays

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5.8
-
 assay at Sorghum bicolor
5.8
-
 assay at Zea mays