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EC Tree
IUBMB Comments The enzyme is involved in ketogluconate metabolism, and catalyses the reaction in vivo in the reverse direction to that shown . It is used in the commercial microbial production of ascorbate. cf. EC 1.1.1.274, 2,5-didehydrogluconate reductase (2-dehydro-D-gluconate-forming).
The taxonomic range for the selected organisms is: Corynebacterium sp. The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
25dkgr-a, 2,5-dkgr a, 2,5-diketo-d-gluconic acid reductase a,
more
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2,5-diketo-D-gluconic acid reductase A
isoform
2,5-diketo-D-gluconate reductase
-
-
2,5-diketo-D-gluconate reductase I
-
isoform
2,5-diketo-D-gluconate reductase II
-
isoform
2,5-diketo-D-gluconic acid reductase
-
-
2,5-diketo-D-gluconic acid reductase A
-
-
2,5-DKGR A
-
-
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2-dehydro-D-gluconate:NADP+ 2-oxidoreductase (2-dehydro-L-gulonate-forming)
The enzyme is involved in ketogluconate metabolism, and catalyses the reaction in vivo in the reverse direction to that shown [1]. It is used in the commercial microbial production of ascorbate. cf. EC 1.1.1.274, 2,5-didehydrogluconate reductase (2-dehydro-D-gluconate-forming).
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2,5-didehydro-D-gluconate + NADPH + H+
2-dehydro-L-gulonate + NADP+
-
-
-
?
2,5-didehydro-D-gluconate + NADPH + H+
2-dehydro-L-gulonate + NADP+
2-dehydro-L-gulonate + NADP+
2,5-didehydro-D-gluconate + NADPH + H+
-
-
-
-
r
5-dehydro-D-fructose + NADPH + H+
? + NADP+
-
-
-
-
?
5-dehydro-D-fructose + NADPH + H+
L-sorbose + NADP+
-
isoforms 2,5-diketo-D-gluconate reductase I and II show 150% and 13% activity, respectively, compared to 2,5-didehydro-D-gluconate
-
-
?
dihydroxyacetone + NADPH + H+
? + NADP+
-
-
-
-
?
additional information
?
-
2,5-didehydro-D-gluconate + NADPH + H+
2-dehydro-L-gulonate + NADP+
-
-
-
-
?
2,5-didehydro-D-gluconate + NADPH + H+
2-dehydro-L-gulonate + NADP+
-
100% activity
-
-
?
2,5-didehydro-D-gluconate + NADPH + H+
2-dehydro-L-gulonate + NADP+
-
a reduction of substrate by NADPH is highly preferred
-
-
r
2,5-didehydro-D-gluconate + NADPH + H+
2-dehydro-L-gulonate + NADP+
-
isoform DKGR B exhibits 66fold higher specific activity toward 2,5-didehydro-D-gluconate than isoform DKGR A
-
-
?
additional information
?
-
-
no activity with 2-dehydro-L-gulonate, 2-dehydro-D-gluconate, 5-dehydro-D-gluconate, D-fructose, and L-sorbose
-
-
?
additional information
?
-
-
the wild type enzyme shows no activity with NADH
-
-
?
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NADH
the enzyme exhibits a preference for NADPH compared to NADH
NADPH
-
-
NADPH
-
the enzyme has a preference for NADPH over NADH
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Cu2+
-
strong inhibition at 0.5 mM
Fe3+
-
strong inhibition at 0.5 mM
NADP+
-
competitive inhibition
Ni2+
-
strong inhibition at 0.5 mM
Zn2+
-
strong inhibition at 0.5 mM
additional information
-
not inhibited by 2-dehydro-L-gulonate. There is no observed effect on activity by 0.5 mM solutions of Mg2+, Mn2+, Ca2+, or Co2+, 14 mM 2-mercaptoethanol, 1 mM dithiothreitol, or 1 mM EDTA
-
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1.8 - 26
2,5-didehydro-D-gluconate
204
2-dehydro-L-gulonate
-
at pH 9.2 and 25°C
155
5-dehydro-D-fructose
-
at pH 6.4 and 25°C
160
dihydroxyacetone
-
at pH 6.4 and 25°C
0.125
NADP+
-
at pH 9.2 and 25°C
1.8
2,5-didehydro-D-gluconate
-
isoform 2,5-diketo-D-gluconate reductase I, at pH 7.0 and 30°C
13.5
2,5-didehydro-D-gluconate
-
isoform 2,5-diketo-D-gluconate reductase II, at pH 7.0 and 30°C
19.78
2,5-didehydro-D-gluconate
-
at pH 6.4 and 25°C
26
2,5-didehydro-D-gluconate
-
at pH 6.4 and 25°C
0.01
NADPH
-
at pH 6.4 and 25°C
0.012
NADPH
-
isoform 2,5-diketo-D-gluconate reductase I, at pH 7.0 and 30°C
0.013
NADPH
-
isoform 2,5-diketo-D-gluconate reductase II, at pH 7.0 and 30°C
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8.3
2,5-didehydro-D-gluconate
-
at pH 6.4 and 25°C
2.2
NADPH
-
mutant enzyme F22Y/S233T/R235S/R238H/A272G, in 100 mM Bis-Tris, pH 7.0, at 25°C
3.3
NADPH
-
mutant enzyme F22Y/A272G, in 100 mM Bis-Tris, pH 7.0, at 25°C
7
NADPH
-
mutant enzyme F22Y/K232G/R235G/R238H/A272G, in 100 mM Bis-Tris, pH 7.0, at 25°C
7.3
NADPH
-
mutant enzyme K232G/R238H, in 100 mM Bis-Tris, pH 7.0, at 25°C
18.3
NADPH
-
mutant enzyme F22Y/K232G/R238H/A272G, in 100 mM Bis-Tris, pH 7.0, at 25°C
18.3
NADPH
-
wild type enzyme, in 100 mM Bis-Tris, pH 7.0, at 25°C
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0.026
NADP+
-
at pH 9.2 and 25°C
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0.0052
-
crude extract, at pH 6.4 and 25°C
0.0276
-
crude extract, at pH 6.4 and 25°C
1.967
-
after 71.03fold purification, at pH 6.4 and 25°C
3.38
-
after 650fold purification, at pH 6.4 and 25°C
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6 - 7
-
isoform 2,5-diketo-D-gluconate reductase I
6 - 7.5
-
isoform 2,5-diketo-D-gluconate reductase II
6.4
-
for the reduction of 2,5-didehydro-D-gluconate
9.2
-
for the oxidation of 2-dehydro-L-gulonate
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5 - 8
-
for the reduction of 2,5-didehydro-D-gluconate
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40
-
isoform 2,5-diketo-D-gluconate reductase I
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4.1
-
isoform 2,5-diketo-D-gluconate reductase II, isoelectric focusing
4.3
-
isoform 2,5-diketo-D-gluconate reductase I, isoelectric focusing
4.4
-
isoelectric focusing
4.7
-
isoelectric focusing
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-
Uniprot
brenda
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-
-
brenda
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29000
-
1 * 29000, isoform 2,5-diketo-D-gluconate reductase I, SDS-PAGE
34000
-
1 * 34000, SDS-PAGE
34000
-
1 * 34000, isoform 2,5-diketo-D-gluconate reductase II, SDS-PAGE
35000
-
gel filtration
35000
-
4 * 35000, SDS-PAGE
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homotetramer
-
4 * 35000, SDS-PAGE
monomer
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1 * 34000, SDS-PAGE
monomer
-
1 * 29000, isoform 2,5-diketo-D-gluconate reductase I, SDS-PAGE
monomer
-
1 * 34000, isoform 2,5-diketo-D-gluconate reductase II, SDS-PAGE
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mutant enzyme F22Y/K232G/R238H/A272G in complex with NADH, hanging drop vapor diffusion method, using 1.5 M lithium sulfate and 0.1 M Na HEPES (pH 7.5), at 22°C
in complex with NADPH, hanging drop vapor diffusion method, using 1 M sodium phosphate, 1 M potassium phosphate, 100 mM HEPES buffer, pH 7.0, at 4°C
-
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F22Y/K232G/R238H/A272G
the mutation enhances binding to NADH, while retaining to a large extent the ability to bind NADPH. The mutant is also more stable and can, therefore, be expected to exhibit greater effective activity at elevated temperatures in comparison to the wild type enzyme
F22Y
-
the mutation causes a 2.5fold decrease in Km for 2,5-didehydro-D-gluconate whereas the value of kcat remains essentially unchanged
F22Y/A272G
-
substrate-binding pocket double mutant with decreased kcat value for NADPH compared to the wild type enzyme
F22Y/K232G/R235G/R238H/A272G
-
mutant with wild type kcat value for NADPH
F22Y/K232G/R235T/R238H/A272G 420
-
mutant with decreased kcat value for NADPH compared to the wild type enzyme
K232G/R238H
-
mutant with decreased kcat value for NADPH compared to the wild type enzyme
K233G
-
the mutant shows decreased NADPH activity and increased NADH activity compared to the wild type enzyme
K233H
-
the mutant shows decreased NADPH activity compared to the wild type enzyme and no NADH activity
K233M
-
the mutant shows decreased NADPH activity and increased NADH activity compared to the wild type enzyme
K233Q
-
the mutant shows wild type NADPH activity and increased NADH activity
K233R
-
the mutant shows decreased NADPH activity compared to the wild type enzyme and no NADH activity
K233S
-
the mutant shows wild type NADPH activity and increased NADH activity compared to the wild type enzyme
K233T
-
the mutant shows wild type NADPH activity and no NADH activity
Q192R
-
the mutation primarily affects the kcat parameter toward the 2,5-didehydro-D-gluconate substrate, increasing its value approximately 2.5fold, whereas Km is relatively unaffected, or increases slightly
R235C
-
the mutant shows wild type NADPH activity and no NADH activity
R235D
-
the mutant shows decreased NADPH activity compared to the wild type enzyme and no NADH activity
R235E
-
the mutant shows decreased NADPH activity compared to the wild type enzyme and no NADH activity
R235G
-
the mutant shows decreased NADPH activity and increased NADH activity compared to the wild type enzyme
R235H
-
the mutant shows wild type NADPH activity and no NADH activity
R235M
-
the mutant shows wild type NADPH activity and no NADH activity
R235N
-
the mutant shows wild type NADPH activity and no NADH activity
R235Q
-
the mutant shows wild type NADPH activity and no NADH activity
R235S
-
the mutant shows wild type NADPH activity and no NADH activity
R235T
-
the mutant shows wild type NADPH activity and increased NADH activity
R235Y
-
the mutant shows reduced NADPH activity compared to the wild type enzyme and no NADH activity
R238D
-
the mutant shows no activity with NADPH and NADH
R238E
-
the mutant shows no activity with NADPH and increased NADH activity compared to the wild type enzyme
R238F
-
the mutant shows wild type NADPH activity and no NADH activity
R238G
-
the mutant shows reduced NADPH activity compared to the wild type enzyme and no NADH activity
R238H
-
the mutant shows wild type NADPH activity and increased NADH activity
R238N
-
the mutant shows reduced NADPH activity and no NADH activity
R238Q
-
the mutant shows reduced NADPH activity compared to the wild type enzyme and no NADH activity
R238Y
-
the mutant shows reduced NADPH activity and increased NADH activity ompared to the wild type enzyme
S233E
-
the mutant shows no activity with NADPH and NADH
S233K
-
the mutant shows no activity with NADPH and NADH
S233M
-
the mutant shows no activity with NADPH and NADH
S233N
-
the mutant shows no activity with NADPH and NADH
S233T
-
the mutant shows wild type NADPH activity and no NADH activity
S233V
-
the mutant shows no activity with NADPH and NADH
V234D
-
the mutant shows wild type NADPH activity and no NADH activity
V234E
-
the mutant shows wild type NADPH activity and no NADH activity
V234I
-
the mutant shows wild type NADPH activity and no NADH activity
V234M
-
the mutant shows wild type NADPH activity and no NADH activity
V234M/R235C
-
the mutant shows wild type NADPH activity and no NADH activity
V234N
-
the mutant shows wild type NADPH activity and no NADH activity
V234Q
-
the mutant shows wild type NADPH activity and no NADH activity
V234S
-
the mutant shows decreased NADPH activity compared to the wild type enzyme and no NADH activity
F22Y/K232G/R238H/A272G
-
mutant with decreased kcat value for NADPH compared to the wild type enzyme
F22Y/K232G/R238H/A272G
-
the mutant exhibits activity with NADH that is more than 2 orders of magnitude higher than that of the wild type enzyme and retains a high level of activity with NADPH
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5 - 7
-
when isoform I is incubated at different pH values for 3 h at 30°C, the remaining activity is about 29% of the original level at pH 5.0, about 65% at pH 6.0 and about 51% at pH 7.0
440303
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0 - 38
-
temperature stability of isoform 2,5-diketo-D-gluconate reductase I is measured by storing the enzyme in 0.1 M Tris-HC1 buffer (pH 7.0) for 1 h. The activity of the enzyme decreases gradually with temperature from 0°C to 27°C, falls rapidly above 27°C, and is lost completely at about 38°C. The remaining activity at 27°C is about 77% of that at 0°C
40 - 80
-
after approximately 10 min of deactivation, enzyme activity attains a constant value, independently of deactivation temperature (40-80°C). This value corresponds to the 30% of native activity
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-70°C, 2 mg/ml enzyme in 20 mM Tris-HC1 (pH 7.5), 6 months, the enzyme remains stable
-
4°C, enzyme solution at pH 6.5-7.5, at least 2 months, the enzyme remains stable
-
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ammonium sulfate precipitation, DEAE-Sepharose column chromatography, and Amicon Matrix Gel Red A gel filtration
-
DEAE cellulose resin column chromatography, gel filtration
-
DEAE-cellulose column chromatography, Cibacron blueF 3GA affinity column chromatography, and and TSK gel filtration
-
DEAE-Sephacel column chromatography and Cibacron blue 3GA column chromatography
-
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expressed in in Acetobacter cerinus strain IFO 3263
-
mutant enzymes are expressed in Escherichia coli JM109 cells
-
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Sonoyama, T.; Kobayashi, K.
Purification and properties of two 2,5-diketo-D-gluconate reductases from a mutant strain derived from Corynebacterium sp
J. Ferment. Technol.
65
311-317
1987
Corynebacterium sp., Corynebacterium sp. SHS 0007
-
brenda
Miller, J.V.; Estell, D.A.; Lazarus, R.A.
Purification and characterization of 2,5-diketo-D-gluconate reductase from Corynebacterium sp
J. Biol. Chem.
262
9016-9020
1987
Corynebacterium sp., Corynebacterium sp. ATCC 31090
brenda
Khurana, S.; Powers, D.B.; Anderson, S.; Blaber, M.
Crystal structure of 2,5-diketo-D-gluconic acid reductase A complexed with NADPH at 2.1 A resolution
Proc. Natl. Acad. Sci. USA
95
6768-6773
1998
Corynebacterium sp.
brenda
Khurana, S.; Sanli, G.; Powers, D.B.; Anderson, S.; Blaber, M.
Molecular modeling of substrate binding in wild-type and mutant Corynebacteria 2,5-diketo-D-gluconate reductases
Proteins
39
68-75
2000
Corynebacterium sp.
brenda
Banta, S.; Swanson, B.A.; Wu, S.; Jarnagin, A.; Anderson, S.
Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A
Protein Eng.
15
131-140
2002
Corynebacterium sp.
brenda
Banta, S.; Swanson, B.A.; Wu, S.; Jarnagin, A.; Anderson, S.
Optimizing an artificial metabolic pathway: engineering the cofactor specificity of Corynebacterium 2,5-diketo-D-gluconic acid reductase for use in vitamin C biosynthesis
Biochemistry
41
6226-6236
2002
Corynebacterium sp.
brenda
Sanli, G.; Banta, S.; Anderson, S.; Blaber, M.
Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase
Protein Sci.
13
504-512
2004
Corynebacterium sp. (P06632)
brenda
Maremonti, M.; Greco Jr., G.; Wichmann, R.
Characterisation of 2,5-diketo-D-gluconic acid reductase from Corynebacterium sp.
Biotechnol. Lett.
18
845-850
1996
Corynebacterium sp.
-
brenda