Information on EC 1.1.1.346 - 2,5-didehydrogluconate reductase (2-dehydro-L-gulonate-forming)

Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)

The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.1.1.346
-
RECOMMENDED NAME
GeneOntology No.
2,5-didehydrogluconate reductase (2-dehydro-L-gulonate-forming)
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2-dehydro-L-gulonate + NADP+ = 2,5-didehydro-D-gluconate + NADPH + H+
show the reaction diagram
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
ketogluconate metabolism
-
-
L-ascorbate biosynthesis VI (engineered pathway)
-
-
ketogluconate metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
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).
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Brevibacterium ketosoreductum
-
-
-
Manually annotated by BRENDA team
Brevibacterium ketosoreductum ATCC21914
-
-
-
Manually annotated by BRENDA team
Escherichia coli BL21-(DE3)
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,5-didehydro-D-gluconate + NADPH + H+
2-dehydro-L-gulonate + NADP+
show the reaction diagram
2,5-didehydro-D-gluconate + NADPH + H+
5-dehydro-D-gluconate + NADP+
show the reaction diagram
Brevibacterium ketosoreductum
-
100% activity
-
-
?
2-dehydro-D-gluconate + NADPH + H+
D-gluconate + NADP+
show the reaction diagram
Brevibacterium ketosoreductum
-
11.8% activity compared to 2,5-didehydro-D-gluconate
-
-
?
2-dehydro-L-gulonate + NADP+
2,5-didehydro-D-gluconate + NADPH + H+
show the reaction diagram
-
-
-
-
r
2-dehydro-L-gulonate + NADPH + H+
L-idonate + NADP+
show the reaction diagram
Brevibacterium ketosoreductum
-
10.8% activity compared to 2,5-didehydro-D-gluconate
-
-
?
5-dehydro-D-fructose + NADPH + H+
? + NADP+
show the reaction diagram
-
-
-
-
?
5-dehydro-D-fructose + NADPH + H+
L-sorbose + NADP+
show the reaction diagram
-
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+
show the reaction diagram
-
-
-
-
?
ethyl 2-acetylpent-4-enoate + NADPH + H+
ethyl (2R)-2-[(1S)-1-hydroxyethyl]pent-4-enoate + NADP+
show the reaction diagram
-
250% activity compared to ethyl 2-methylacetoacetate
-
-
?
ethyl 2-ethyl-3-oxobutanoate + NADPH + H+
ethyl (2R,3S)-2-ethyl-3-hydroxybutanoate + NADP+
show the reaction diagram
-
120% activity compared to ethyl 2-methylacetoacetate
-
-
?
ethyl 2-methylacetoacetate + NADH + H+
ethyl (2R)-methyl-(3S)-hydroxybutanoate + NAD+
show the reaction diagram
-
7% activity with NADH compared to NADPH
-
-
?
ethyl 2-methylacetoacetate + NADPH + H+
ethyl (2R)-methyl-(3S)-hydroxybutanoate + NADP+
show the reaction diagram
-
-
-
-
?
ethyl acetoacetate + NADPH + H+
ethyl (3S)-3-hydroxybutanoate + NADP+
show the reaction diagram
-
53% activity compared to ethyl 2-methylacetoacetate
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADH
the enzyme exhibits a preference for NADPH compared to NADH
NADPH
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
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
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.8 - 26
2,5-didehydro-D-gluconate
204
2-dehydro-L-gulonate
-
at pH 9.2 and 25C
155
5-dehydro-D-fructose
-
at pH 6.4 and 25C
160
dihydroxyacetone
-
at pH 6.4 and 25C
3.1
ethyl 2-methylacetoacetate
-
at pH 7.0 and 30C
0.125
NADP+
-
at pH 9.2 and 25C
0.01 - 0.013
NADPH
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8.3
2,5-didehydro-D-gluconate
Corynebacterium sp.
-
at pH 6.4 and 25C
2.6
ethyl 2-methylacetoacetate
Escherichia coli
-
at pH 7.0 and 30C
2.2 - 18.3
NADPH
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.026
NADP+
-
at pH 9.2 and 25C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0052
-
crude extract, at pH 6.4 and 25C
0.0276
-
crude extract, at pH 6.4 and 25C
0.07
Brevibacterium ketosoreductum
-
crude extract, at pH 6.4 and 30C
1.967
-
after 71.03fold purification, at pH 6.4 and 25C
3.38
-
after 650fold purification, at pH 6.4 and 25C
6.3
-
purified enzyme, at pH 7.0 and 30C
22.2
Brevibacterium ketosoreductum
-
after 317fold purification, at pH 6.4 and 30C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
Brevibacterium ketosoreductum
-
-
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
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 8
-
for the reduction of 2,5-didehydro-D-gluconate
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
-
isoform 2,5-diketo-D-gluconate reductase I
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
5.9
-
isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Corynebacterium sp. (strain ATCC 31090)
Corynebacterium sp. (strain ATCC 31090)
Corynebacterium sp. (strain ATCC 31090)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30000
gel filtration
35000
-
gel filtration
72000
Brevibacterium ketosoreductum
-
gel filtration
140000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
homotetramer
-
4 * 35000, SDS-PAGE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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 4C
-
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 22C
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 7
-
when isoform I is incubated at different pH values for 3 h at 30C, 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
5.2 - 10
Brevibacterium ketosoreductum
-
after 2 h incubation at 30C, the remaining activity is about 35% of the original level at pH 8.4, and no remaining activity is observed below pH 5.1 and 10.0. The enzyme is stable from pH 6.0 to 7.5
286278
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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 0C to 27C, falls rapidly above 27C, and is lost completely at about 38C. The remaining activity at 27C is about 77% of that at 0C
40 - 80
-
after approximately 10 min of deactivation, enzyme activity attains a constant value, independently of deactivation temperature (40-80C). This value corresponds to the 30% of native activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, 2 mg/ml enzyme in 20 mM Tris-HC1 (pH 7.5), 6 months, the enzyme remains stable
-
4C, enzyme solution at pH 6.5-7.5, at least 2 months, the enzyme remains stable
-
4C, potassium buffer, several weeks, no loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, DEAE Toyopearl column chromatography and phenyl Toyopearl column chromatography, Blue Sepharose column chromatography and Sephacryl S-200 gel filtration
Brevibacterium ketosoreductum
-
ammonium sulfate precipitation, DEAE-Sepharose column chromatography, and Amicon Matrix Gel Red A gel filtration
-
ammonium sulfate precipitation, DEAE-Sepharose column chromatography, Matrex Red-A column chromatography, Sephacryl S-200 gel filtration, and hypatite C cholumn chromatography
-
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
-
Ni-NTA column chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in in Acetobacter cerinus strain IFO 3263
-
mutant enzymes are expressed in Escherichia coli JM109 cells
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
F22Y/K232G/R238H/A272G
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