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Literature summary extracted from

  • Bubner, P.; Klimacek, M.; Nidetzky, B.
    Structure-guided engineering of the coenzyme specificity of Pseudomonas fluorescens mannitol 2-dehydrogenase to enable efficient utilization of NAD(H) and NADP(H) (2008), FEBS Lett., 582, 233-237.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.1.1.67 additional information no activation of M2DH by bovine serum albumin Pseudomonas fluorescens

Application

EC Number Application Comment Organism
1.1.1.67 industry redox balancing between the intracellular NADP(H) and NAD(H) based on NAD(P)(H)-dependent interconversion of mannitol and fructose by M2DH may be a useful strategy of metabolic engineering Pseudomonas fluorescens

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.67 expressed in Escherichia coli Pseudomonas fluorescens
1.1.1.67 expression of wild-type and mutant enzymes in Escherichia coli strain JM109 Pseudomonas fluorescens
1.1.1.138 expression of mutant enzymes in Escherichia coli strain JM109 Pseudomonas fluorescens

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.67 D69A site-directed mutagenesis, the mutant shows an altered cofactor specificity compared to the wild-type enzyme, which is switched to NADP(H), EC 1.1.1.138, NADP(H) is equally utilized as NAD(H) Pseudomonas fluorescens
1.1.1.67 D69A utilizes NAD(H) and NADP(H) with similar catalytic efficiencies. Uses NADP(H) almost as well as wild-type enzyme uses NAD(H) Pseudomonas fluorescens
1.1.1.67 E68K site-directed mutagenesis, the mutant shows an altered cofactor specificity compared to the wild-type enzyme, which is switched to NADP(H), EC 1.1.1.138, NADP(H) is preferred by 10fold over NAD(H) Pseudomonas fluorescens
1.1.1.67 E68K/D69A shows about a 10fold preference for NADP(H) over NAD(H), accompanied by a small decrease in catalytic efficiency for NAD(H)-dependent reactions as compared to wild-type enzyme Pseudomonas fluorescens
1.1.1.138 D69A site-directed mutagenesis, the mutant shows an altered cofactor specificity compared to the wild-type enzyme, cf. EC 1.1.1.67, which is switched to NADP(H), NADP(H) is equally utilized as NAD(H) Pseudomonas fluorescens
1.1.1.138 E68K site-directed mutagenesis, the mutant shows an altered cofactor specificity compared to the wild-type enzyme, cf. EC 1.1.1.67, which is switched to NADP(H), NADP(H) is preferred by 10fold over NAD(H) Pseudomonas fluorescens

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.1.1.67 additional information no inhibition of M2DH by bovine serum albumin Pseudomonas fluorescens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.67 additional information
-
 additional information steady-state kinetic analysis, recombinant wild-type and mutant enzymes, overview Pseudomonas fluorescens
1.1.1.138 additional information
-
 additional information steady-state kinetic analysis, recombinant mutant enzymes, overview Pseudomonas fluorescens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.67 D-mannitol + NAD+ Pseudomonas fluorescens wild-type enzyme D-fructose + NADH + H+
-
 r
1.1.1.138 D-mannitol + NADP+ Pseudomonas fluorescens
-
 D-fructose + NADPH + H+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.67 Pseudomonas fluorescens O08355
-
-
1.1.1.138 Pseudomonas fluorescens
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.67 mutants purified to apparent homogeneity Pseudomonas fluorescens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.67 D-mannitol + NAD(P)+
-
 Pseudomonas fluorescens D-fructose + NAD(P)H + H+
-
 r
1.1.1.67 D-mannitol + NAD+ wild-type enzyme Pseudomonas fluorescens D-fructose + NADH + H+
-
 r
1.1.1.138 D-mannitol + NADP+
-
 Pseudomonas fluorescens D-fructose + NADPH + H+
-
 r
1.1.1.138 D-mannitol + NADP+ only the mutant enzymes D69A amd E68K, the wild-type enzyme utilizes NAD(H) as cofactor, EC 1.1.1.67 Pseudomonas fluorescens D-fructose + NADPH + H+
-
 r

Synonyms

EC Number Synonyms Comment Organism
1.1.1.67 M2DH
-
 Pseudomonas fluorescens
1.1.1.67 mannitol 2-dehydrogenase
-
 Pseudomonas fluorescens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.67 25
-
 assay at, both reaction directions Pseudomonas fluorescens
1.1.1.138 25
-
 assay at, both reaction directions Pseudomonas fluorescens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.67 3
-
 NAD(P)H wild-type Pseudomonas fluorescens
1.1.1.67 4.6
-
 NADH mutant D69A/D69A Pseudomonas fluorescens
1.1.1.67 12
-
 NAD(P)H mutant D69A/D69A Pseudomonas fluorescens
1.1.1.67 15
-
 NADH mutant D69A Pseudomonas fluorescens
1.1.1.67 24
-
 NAD(P)H mutant D69A Pseudomonas fluorescens
1.1.1.67 26
-
 NADH wild-type Pseudomonas fluorescens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.67 8
-
 assay at, both reaction directions Pseudomonas fluorescens
1.1.1.138 8
-
 assay at, both reaction directions Pseudomonas fluorescens

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.67 NAD+
-
 Pseudomonas fluorescens
1.1.1.67 NAD+ binding structure, the carboxylate group of Asp69 forms a bifurcated hydrogen bond with the 2' and 3' hydroxyl groups of the adenosine of NAD+ and contributes to the 400fold preference of the enzyme for NAD+ as compared to NADP+, overview Pseudomonas fluorescens
1.1.1.67 NADH
-
 Pseudomonas fluorescens
1.1.1.67 NADP+ 400fold preference of the enzyme for NAD+ as compared to NADP+ Pseudomonas fluorescens
1.1.1.138 NADP+
-
 Pseudomonas fluorescens
1.1.1.138 NADPH
-
 Pseudomonas fluorescens