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Information on EC 1.1.1.274 - 2,5-didehydrogluconate reductase (2-dehydro-D-gluconate-forming) and Organism(s) Corynebacterium sp. and UniProt Accession P06632

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EC Tree
IUBMB Comments
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).
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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
2,5-dkg reductase, 2,5-dkg, 2,5-dkgr, 2,5-diketo-d-gluconate reductase, beta-keto ester reductase, yqhe reductase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2,5-diketo-D-gluconic acid reductase
-
2,5-diketo-D-gluconate reductase
-
-
-
-
YqhE reductase
-
-
-
-
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 mechanism of wild-type and F22Y/K232G/R238H/A272G mutant enzyme
2-dehydro-D-gluconate + NADP+ = 2,5-didehydro-D-gluconate + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
redox reaction
-
-
-
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oxidation
-
-
-
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reduction
-
-
-
-
PATHWAY SOURCE
PATHWAYS
-
-, -
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
-
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 + NADH
2-keto-L-gulonate + NAD+
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 + 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
5-keto-D-fructose + NADPH
L-sorbose + NADP+
show the reaction diagram
dihydroxyacetone + NADPH
glycerol + NADP+
show the reaction diagram
additional information
?
-
isozyme A is more stable than isozyme B but less active
-
-
?
NATURAL SUBSTRATE
NATURAL 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
2-oxo-L-gulonic acid + NADP+
show the reaction diagram
step in the biosynthesis of L-ascorbic acid
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADH
F22Y/K232G/R238H/A272G mutant enzyme
NADPH
preferred cofactor of the wild-type enzyme
additional information
cofactor binding structure of wild-type and F22Y/K232G/R238H/A272G mutant enzyme, involved Lys232, Ala272, and Arg238
-
INHIBITOR
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
2 - 8.8
NADH
0.0123 - 150
2,5-didehydro-D-gluconate
155
5-keto-D-fructose
-
-
160
dihydroxyacetone
-
-
2.2
glycerol
-
-
0.66 - 5.2
NADH
0.01 - 13
NADPH
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8.33
2,5-didehydro-D-gluconate
-
-
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
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 7
-
isoenzyme I
6 - 7.5
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isoenzyme II
6.4
-
reduction of 2,5-didehydro-D-gluconate
9.2
-
oxidation of 2-keto-L-gulonate
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 9.3
-
reduction of 2,5-didehydro-D-gluconate
8 - 10.3
-
oxidation of 2-keto-L-gulonate
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
29000
-
isoenzyme I
29992
-
1 * 29992, deduced from nucleotide sequence
34000
35000
-
gel filtration
SUBUNIT
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
isoenzyme A, hanging-drop vapour diffusion, X-ray structure of complex with NADPH, 2.1 A resolution
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A272G
mutation increases Km and kcat compared to the wild-type enzyme
F22Y
mutation reduces Km and increases kcat by 50% compared to the wild-type enzyme
F22Y/A272G
increased activity compared to the wild-type enzyme, substrate inhibition at substrate concentrations above 17.5 mM
F22Y/K232G/R238H/A272G
mutant shows a higher activity with NADH compared to the wild-type enzyme
K232
isoenzyme A, designed to improve the ability to use NADH as cofactor
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
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
F22Y
-
isoenzyme A, similar kcat as wild-type
F22Y/A272G
-
isoenzyme A, reduced kcat for NADH
F22Y/K232G/R235G/R238E/A272G
-
isoenzyme A, increase in kcat for NADH
F22Y/K232G/R235G/R238H/A272G
-
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
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isoenzyme A, increase in kcat forNADH
F22Y/S232T/R235S/R238H/A272G
-
isoenzyme A, increase in kcat for NADH
K232G/R238H
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isoenzyme A, increase in kcat for NADH
Q192R
-
isoenzyme A, 2.5fold increase in kcat
additional information
mutagenesis of 3 amino acids in the cofactor-binding pocket, mutations lead to higher activity with NADH as cofactor
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
-70°C, 20 mM Tris-HCl, pH 7.5, over 6 months, no loss of activity
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4°C, pH 6.5-7.5, at least 2 months, no loss of activity
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant isoenzyme A, Red A dye-affinity, anion exchange, gel filtration
DEAE-cellulose, Cibacron blue F3GA agarose, gel filtration
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recombinant isoenzyme A
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of isoenzyme A in Escherichia coli
expresssion of several isoenzyme A mutants in Escherichia coli
isoenzyme A, expression in Acetobacter cerinus
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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
enzyme can be used in the industrial production of vitamin C
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.
-
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.
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
Ji, A.; Gao, P.
Substrate selectivity of Gluconobacter oxydans for production of 2,5-diketo-D-gluconic acid and synthesis of 2-keto-L-gulonic acid in a multienzyme system
Appl. Biochem. Biotechnol.
94
213-223
2001
Corynebacterium sp.
Manually annotated by BRENDA team
Sanli, G.; Blaber, M.
Structural assembly of the active site in an aldo-keto reductase by NADPH cofactor
J. Mol. Biol.
309
1209-1218
2001
Corynebacterium sp. (P06632)
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. (P06632)
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
Banta, S.; Anderson, S.
Verification of a novel NADH-binding motif: combinatorial mutagenesis of three amino acids in the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase
J. Mol. Evol.
55
623-631
2002
Corynebacterium sp. (P06632)
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