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Information on EC 1.1.1.346 - 2,5-didehydrogluconate reductase (2-dehydro-L-gulonate-forming) and Organism(s) Corynebacterium sp. and UniProt Accession P06632

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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).
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This record set is specific for:
Corynebacterium sp.
UNIPROT: P06632
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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
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2,5-diketo-D-gluconic acid reductase A
isoform
2,5-DKGRA
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-DKG reductase
-
-
2,5-DKGR A
2,5-DKGR B
-
isoform
PATHWAY SOURCE
PATHWAYS
-
-, -
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).
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+
2-dehydro-L-gulonate + NADP+
show the reaction diagram
2-dehydro-L-gulonate + NADP+
2,5-didehydro-D-gluconate + NADPH + H+
show the reaction diagram
-
-
-
-
r
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
-
-
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADH
the enzyme exhibits a preference for NADPH compared to NADH
NADPH
INHIBITOR
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 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
0.01 - 0.013
NADPH
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8.3
2,5-didehydro-D-gluconate
-
at pH 6.4 and 25°C
2.2 - 18.3
NADPH
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.026
NADP+
-
at pH 9.2 and 25°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
4.7
-
isoelectric focusing
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
Uniprot
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
140000
-
gel filtration
29000
-
1 * 29000, isoform 2,5-diketo-D-gluconate reductase I, SDS-PAGE
34000
35000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
-
4 * 35000, SDS-PAGE
monomer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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 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
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-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
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in in Acetobacter cerinus strain IFO 3263
-
mutant enzymes are expressed in Escherichia coli JM109 cells
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
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
-
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
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.
Manually annotated by BRENDA team
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.
Manually annotated by BRENDA team
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.
Manually annotated by BRENDA team
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.
Manually annotated by BRENDA team
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)
Manually annotated by BRENDA team
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.
-
Manually annotated by BRENDA team