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Literature summary extracted from

  • Sanli, G.; Banta, S.; Anderson, S.; Blaber, M.
    Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase (2004), Protein Sci., 13, 504-512.
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
1.1.1.274 biotechnology enzyme is a target for the construction of a NADH-utilizing mutant strain in the industrial production of vitamin C Corynebacterium sp.
1.1.1.274 synthesis enzyme can be used in the industrial production of vitamin C Corynebacterium sp.

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.274 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 Corynebacterium sp.
1.1.1.346 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 Corynebacterium sp.

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.274 A272G mutation increases Km and kcat compared to the wild-type enzyme Corynebacterium sp.
1.1.1.274 F22Y mutation reduces Km and increases kcat by 50% compared to the wild-type enzyme Corynebacterium sp.
1.1.1.274 F22Y/A272G increased activity compared to the wild-type enzyme, substrate inhibition at substrate concentrations above 17.5 mM Corynebacterium sp.
1.1.1.274 F22Y/K232G/R238H/A272G mutant shows a higher activity with NADH compared to the wild-type enzyme Corynebacterium sp.
1.1.1.346 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 Corynebacterium sp.

General Stability

EC Number General Stability Organism
1.1.1.274 isozyme A is more stable than isozyme B but less active Corynebacterium sp.

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.1.1.274 2,5-didehydro-D-gluconate substrate inhibition of isozyme B, not of isozyme A, at high concentrations Corynebacterium sp.

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.1.1.274 29000
-
 x * 29000 Corynebacterium sp.

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.274 2,5-didehydro-D-gluconate + NADPH Corynebacterium sp. step in the biosynthesis of L-ascorbic acid 2-oxo-L-gulonic acid + NADP+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.274 Corynebacterium sp. P06632 2 isozymes A and B
-
1.1.1.346 Corynebacterium sp. P06632
-
-

Reaction

EC Number Reaction Comment Organism Reaction ID
1.1.1.274 2-dehydro-D-gluconate + NADP+ = 2,5-didehydro-D-gluconate + NADPH + H+ reaction mechanism of wild-type and F22Y/K232G/R238H/A272G mutant enzyme Corynebacterium sp.
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.274 2,5-didehydro-D-gluconate + NADPH step in the biosynthesis of L-ascorbic acid Corynebacterium sp. 2-oxo-L-gulonic acid + NADP+
-
 ?
1.1.1.274 2,5-didehydro-D-gluconate + NADPH i.e. 2,5-diketo-D-gluconic acid or 2,5-DKG, stereospecific reaction Corynebacterium sp. 2-oxo-L-gulonic acid + NADP+ i.e. 2-keto-L-gulonic acid or 2-KLG, product is a precursor for L-ascorbic acid ?
1.1.1.274 additional information isozyme A is more stable than isozyme B but less active Corynebacterium sp. ?
-
 ?
1.1.1.346 2,5-didehydro-D-gluconate + NADPH + H+
-
 Corynebacterium sp. 2-dehydro-L-gulonate + NADP+
-
 ?

Subunits

EC Number Subunits Comment Organism
1.1.1.274 ? x * 29000 Corynebacterium sp.

Synonyms

EC Number Synonyms Comment Organism
1.1.1.274 2,5-diketo-D-gluconic acid reductase
-
 Corynebacterium sp.
1.1.1.274 2,5-DKGR
-
 Corynebacterium sp.
1.1.1.346 2,5-diketo-D-gluconic acid reductase A isoform Corynebacterium sp.
1.1.1.346 2,5-DKGRA isoform Corynebacterium sp.

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.1.1.274 additional information
-
 thermodynamic stability study of wild-type and F22Y/K232G/R238H/A272G mutant enzyme Corynebacterium sp.

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.274 additional information cofactor binding structure of wild-type and F22Y/K232G/R238H/A272G mutant enzyme, involved Lys232, Ala272, and Arg238 Corynebacterium sp.
1.1.1.274 NADH F22Y/K232G/R238H/A272G mutant enzyme Corynebacterium sp.
1.1.1.274 NADPH preferred cofactor of the wild-type enzyme Corynebacterium sp.
1.1.1.346 NADH the enzyme exhibits a preference for NADPH compared to NADH Corynebacterium sp.