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
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
ketogluconate metabolism
-
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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
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
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
additional information
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
-
-
-
-
?
2-dehydro-D-gulonate + NADP+
2,5-didehydro-D-gluconate + NADPH + H+
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-glucuronic acid + NADPH
? + NADP+
show the reaction diagram
-
-
-
-
?
D-xylose + NADPH
? + NADP+
show the reaction diagram
-
-
-
-
?
dihydroxyacetone + NADPH
glycerol + NADP+
show the reaction diagram
DL-glyceraldehyde + NADPH + H+
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
-
-
-
?
2-dehydro-D-gulonate + NADP+
2,5-didehydro-D-gluconate + NADPH + H+
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 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+
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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
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-
160
dihydroxyacetone
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3.1
ethyl 2-methylacetoacetate
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pH 7.0, 30C, with a cofactor level 20fold higher than the Km value
3.1
ethyl-2-methylacetoacetate
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-
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
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2.6
ethyl 2-methylacetoacetate
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pH 7.0, 30C
2.6
ethyl-2-methylacetoacetate
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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+
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-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.01
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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
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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
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YafB, 1 mM benzaldehyde as substrate
3.88
-
YqhE, 1 mM benzaldehyde as substrate
6.53
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YafB, 1 mM 4-nitrobenzaldehyde as substrate
10.3
-
YqhE, 1 mM 4-nitrobenzaldehyde as substrate
14.9
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YafB, 1 mM methylglyoxal as substrate
21
-
YqhE, 1 mM methylglyoxal as substrate
39.1
-
YafB, 1 mM phenylglyoxal as substrate
64.4
-
YqhE, 1 mM phenylglyoxal as substrate
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 7
-
isoenzyme I
6 - 7.5
-
isoenzyme II
6.4
-
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
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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
29992
-
1 * 29992, deduced from nucleotide sequence
30000
isoenzymes A and B, gel filtration
31800
-
1 * 31800, SDS-PAGE
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 His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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 in Escherichia coli
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 CTATCC11996_22452, genetic organization, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis and tree, recombinant overexpression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
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
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the enzyme expression is significantly induced by estrone, estradiol, and methyltestosterone, and slightly by progesterone, but not by testosterone
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
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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
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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
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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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