Information on EC 1.1.1.274 - 2,5-didehydrogluconate reductase (2-dehydro-D-gluconate-forming)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.1.1.274
-
RECOMMENDED NAME
GeneOntology No.
2,5-didehydrogluconate reductase (2-dehydro-D-gluconate-forming)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2-dehydro-D-gluconate + NADP+ = 2,5-didehydro-D-gluconate + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
ketogluconate metabolism
-
-
L-ascorbate biosynthesis VI (engineered pathway)
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ketogluconate metabolism
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-
SYSTEMATIC NAME
IUBMB Comments
2-dehydro-D-gluconate:NADP+ 2-oxidoreductase (2-dehydro-D-gluconate-forming)
The enzyme is involved in the catabolism of 2,5-didehydrogluconate. cf. EC 1.1.1.346, 2,5-didehydrogluconate reductase (2-dehydro-L-gulonate-forming).
CAS REGISTRY NUMBER
COMMENTARY hide
95725-95-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene dkr
UniProt
Manually annotated by BRENDA team
strain K12
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
2,5-diketo-D-gluconic acid reductase catalyses the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid, a direct precursor (lactone) of L-ascorbic acid (vitamin C). This reaction is an essential step in the biocatalytic production of the food supplement vitamin C from D-glucose or D-gluconic acid
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,2-naphthoquinone + NADPH
? + NADP+
show the reaction diagram
-
-
-
-
?
2,5-didehydro-D-gluconate + NADH
2-keto-L-gulonate + NAD+
show the reaction diagram
2,5-didehydro-D-gluconate + NADPH
2-keto-L-gulonate + NADP+
show the reaction diagram
2,5-didehydro-D-gluconate + NADPH
2-oxo-L-gulonic acid + NADP+
show the reaction diagram
2,5-didehydro-D-gluconate + NADPH + H+
2-dehydro-D-gluconate + NADP+
show the reaction diagram
-
-
-
?
2,5-diketo-D-gluconate + NADPH
? + NADP+
show the reaction diagram
-
-
-
-
?
2-carboxybenzaldehyde + NADPH
2-hydroxybenzoate + NADP+
show the reaction diagram
-
-
-
-
?
3-nitrobenzaldehyde + NADPH
3-nitrobenzyl alcohol + NADP+
show the reaction diagram
-
-
-
-
?
4-nitrobenzaldehyde + NADPH
4-nitrobenzyl alcohol + NADP+
show the reaction diagram
5-keto-D-fructose + NADPH
L-sorbose + NADP+
show the reaction diagram
benzaldehyde + NADPH
benzyl alcohol + NADP+
show the reaction diagram
D,L-glyceraldehyde + NADPH
glycerol + NADP+
show the reaction diagram
-
-
-
-
?
D-glucuronic acid + NADPH
? + NADP+
show the reaction diagram
-
-
-
-
?
D-xylose + NADPH
? + NADP+
show the reaction diagram
dihydroxyacetone + NADPH
glycerol + NADP+
show the reaction diagram
ethyl 2-ethylacetoacetate + NADPH
ethyl (2R)-ethyl-(3S)-hydroxybutanoate + NADP+
show the reaction diagram
-
stereospecific reaction
-
-
?
ethyl 2-methylacetoacetate + NADPH
ethyl (2R)-methyl-(3S)-hydroxybutanoate + NADP+
show the reaction diagram
-
stereospecific reaction
-
-
?
ethyl 2-methylacetoacetate + NADPH + H+
ethyl (2R)-methyl-(3S)-hydroxybutanoate + NADP+
show the reaction diagram
ethyl acetoacetate + NADPH
ethyl (3S)-hydroxybutanoate + NADP+
show the reaction diagram
-
stereospecific reaction
-
-
?
ethyl-(2R)-allylacetoacetate + NADPH
?
show the reaction diagram
-
250% of activity with ethyl-2-methylacetoacetate
-
-
?
ethyl-(2R)-ethylacetoacetate + NADPH
ethyl-(2R)-ethyl-(3S)-hydroxybutanoate + NADP+
show the reaction diagram
ethylacetoacetate + NADPH
ethyl-(3S)-hydroxybutanoate + NADP+
show the reaction diagram
methylglyoxal + NADPH
? + NADP+
show the reaction diagram
phenylglyoxal + NADPH
? + NADP+
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
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
2-oxo-L-gulonic acid + NADP+
show the reaction diagram
P06632
step in the biosynthesis of L-ascorbic acid
-
-
?
2,5-didehydro-D-gluconate + NADPH + H+
2-dehydro-D-gluconate + NADP+
show the reaction diagram
H9CWC0
-
-
-
?
ethyl 2-ethylacetoacetate + NADPH
ethyl (2R)-ethyl-(3S)-hydroxybutanoate + NADP+
show the reaction diagram
-
stereospecific reaction
-
-
?
ethyl 2-methylacetoacetate + NADPH
ethyl (2R)-methyl-(3S)-hydroxybutanoate + NADP+
show the reaction diagram
-
stereospecific reaction
-
-
?
ethyl acetoacetate + NADPH
ethyl (3S)-hydroxybutanoate + NADP+
show the reaction diagram
-
stereospecific reaction
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADP+
NADPH
additional information
cofactor binding structure of wild-type and F22Y/K232G/R238H/A272G mutant enzyme, involved Lys232, Ala272, and Arg238
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,5-didehydro-D-gluconate
substrate inhibition of isozyme B, not of isozyme A, at high concentrations
Cu2+
-
0.5 mM, strong inhibition
Fe3+
-
0.5 mM, strong inhibition
NADP+
-
competitive product inhibition
Ni2+
-
0.5 mM, strong inhibition
Zn2+
-
0.5 mM, strong inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0123 - 150
2,5-didehydro-D-gluconate
155
5-keto-D-fructose
-
-
160
dihydroxyacetone
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-
3.1
ethyl 2-methylacetoacetate
2.2
glycerol
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-
2.05 - 2.46
methylglyoxal
0.66 - 8.8
NADH
0.01 - 13
NADPH
additional information
additional information
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-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8.33
2,5-didehydro-D-gluconate
Corynebacterium sp.
-
-
2.6
ethyl 2-methylacetoacetate
27.62 - 81
methylglyoxal
1.23 - 68.3
NADH
2.17 - 18.3
NADPH
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.026
NADP+
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.01
-
YafB, 10 mM D-xylose as substrate; YafB, 1 mM 2-carboxybenzaldehyde as substrate; YqhE, 1 mM 2-carboxybenzaldehyde as substrate
0.0153
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YafB, 10 mM D-glucuronic acid as substrate
0.0247
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YqhE, 10 mM D-glucuronic acid as substrate
0.0401
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YafB, 1 mM 2,5-diketo-D-gluconate as substrate
0.0424
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YafB, 0.1 mM 1,2-naphthoquinone as substrate
0.0519
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YqhE, 1 mM 2,5-diketo-D-gluconate as substrate
0.0949
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YqhE, 10 mM D-xylose as substrate
0.127
-
YqhE, 0.1 mM 1,2-naphthoquinone as substrate
1.36
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YafB, 1 mM DL-glyceraldehyde as substrate
2.21
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YqhE, 1 mM DL-glyceraldehyde as substrate
2.23
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YafB, 1 mM 3-nitrobenzaldehyde as substrate
2.5
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YqhE, 1 mM 3-nitrobenzaldehyde as substrate
2.79
-
YafB, 1 mM benzaldehyde as substrate
3.88
-
YqhE, 1 mM benzaldehyde as substrate
6.53
-
YafB, 1 mM 4-nitrobenzaldehyde as substrate
10.3
-
YqhE, 1 mM 4-nitrobenzaldehyde as substrate
14.9
-
YafB, 1 mM methylglyoxal as substrate
21
-
YqhE, 1 mM methylglyoxal as substrate
39.1
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YafB, 1 mM phenylglyoxal as substrate
64.4
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YqhE, 1 mM phenylglyoxal as substrate
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 7
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isoenzyme I
6 - 7.5
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isoenzyme II
6.4
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reduction of 2,5-didehydro-D-gluconate
7.5
isoenzyme A
9.2
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oxidation of 2-keto-L-gulonate
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 9.3
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reduction of 2,5-didehydro-D-gluconate
8 - 10.3
-
oxidation of 2-keto-L-gulonate
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
assay at
PDB
SCOP
CATH
ORGANISM
UNIPROT
Rhizobium meliloti (strain 1021)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
29000
-
isoenzyme I
30000
isoenzymes A and B, gel filtration
34000
-
isoenzyme II
35000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
10 mg/ml purified recombinant F22Y/K232G/R238H/A272G mutant enzyme in complex with NADH in 25 mM Tris-HCl, pH 7.5, 1 mM NADH, hanging drop vapour diffusion method, room temperature, equal volumes, 0.005 ml each, of protein and crystallization solution against 1 ml reservoir crystallization solution containing 1.5 M lithium sulfate, 0.1 M Na-HEPES, pH 7.5, 7-10 days, X-ray diffraction structure determination and analysis at 2.0 A resolution, molecular replacment, molecular modeling of substrate and cofactor binding
isoenzyme A, hanging drop-vapour diffusion, 1.9 A resolution of apo enzyme
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isoenzyme A, hanging-drop vapour diffusion, X-ray structure of complex with NADPH, 2.1 A resolution
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
thermodynamic stability study of wild-type and F22Y/K232G/R238H/A272G mutant enzyme
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
isozyme A is more stable than isozyme B but less active
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, 20 mM Tris-HCl, pH 7.5, over 6 months, no loss of activity
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4C, pH 6.5-7.5, at least 2 months, no loss of activity
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4C, purified enzyme, 400 mM potassium phoshate buffer, pH 7.0, several weeks without appreciable loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate, DEAE-Sepharose, Matrix Red-A, Sephacryl S-200, hypatite C; native enzyme from strain BL21(DE3), 730fold to homogeneity by ammonium sulfate fractionation, DEAE ion exchange and Red-A affinity chromatography, ultrafiltration, hypatite C-resin chromatography, and a second ultrafiltration step
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DEAE-cellulose, Cibacron blue F3GA agarose, gel filtration
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DEAE-Sepharose column chromatography and Superdex 200 gel filtration
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recombinant isoenzyme A
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recombinant isoenzyme A, Red A dye-affinity, anion exchange, gel filtration
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recombinant isoenzymes A and B, affinity chitin-binding tag
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of isoenzyme A in Escherichia coli
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expresssion of several isoenzyme A mutants in Escherichia coli
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gene dkr, recombinant expression in Lactococcus lactis subsp. cremoris strains MG1363 or NZ3900 and Lactobacillus plantarum strain WCFS1 using food-grade vectors
gne yqhE, DNA and amino acid sequence determination and analysis
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isoenzyme A, expression in Acetobacter cerinus
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overexpression of isoenzymes A and B
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A272G
mutation increases Km and kcat compared to the wild-type enzyme
F22Y/A272G
F22Y/K232G/R235G/R238E/A272G
-
isoenzyme A, increase in kcat for NADH
F22Y/K232G/R235G/R238H/A272G
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isoenzyme A, increase in kcat for NADH
F22Y/K232G/R235T/R238E/A272G
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isoenzyme A, 24fold increase in kcat for NADH
F22Y/K232G/R238H/A272G
F22Y/S232T/R235S/R238H/A272G
-
isoenzyme A, increase in kcat for NADH
K232
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isoenzyme A, designed to improve the ability to use NADH as cofactor
K232G/R238H
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isoenzyme A, increase in kcat for NADH
K232Q
-
isoenzyme A, designed to improve the ability to use NADH as cofactor
K232S
-
isoenzyme A, designed to improve the ability to use NADH as cofactor
Q192R
-
isoenzyme A, 2.5fold increase in kcat
R235G
-
isoenzyme A, designed to improve the ability to use NADH as cofactor
R235T
-
isoenzyme A, designed to improve the ability to use NADH as cofactor
R238E
-
isoenzyme A, designed to improve the ability to use NADH as cofactor
R238H
-
isoenzyme A, designed to improve the ability to use NADH as cofactor, 7fold higher activity with NADH than wild-type
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
enzyme is a target for the construction of a NADH-utilizing mutant strain in the industrial production of vitamin C
synthesis
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