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Literature summary extracted from

  • Klimacek, M.; Nidetzky, B.
    The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites (2010), Biochem. J., 425, 455-463.
    View publication on PubMed

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.67 N191A the rate constants for the overall hydride transfer to and from C-2 of mannitol are selectively slowed, between 540- and 2700fold. Partial disruption of the oxyanion hole in the single-site mutant causes an upshift, by about 1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme–NAD+-mannitol complex Pseudomonas fluorescens
1.1.1.67 N191A/N300A the rate constants for the overall hydride transfer to and from C-2 of mannitol are selectively slowed, with additive effects in the double mutant Pseudomonas fluorescens
1.1.1.67 N191L the rate constants for the overall hydride transfer to and from C-2 of mannitol are selectively slowed, between 540- and 2700fold. Partial disruption of the oxyanion hole in the single-site mutant causes an upshift, by about 1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme–NAD+-mannitol complex Pseudomonas fluorescens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.67 0.0033
-
 NADH mutant N191L, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 0.017
-
 NADH mutant N191A, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 0.023
-
 NADH mutant N191A/N300A, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 0.055
-
 NAD+ mutant N191L, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 0.067
-
 NADH wild-type, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 0.093
-
 NAD+ wild-type, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 0.24
-
 D-fructose wild-type, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 0.31
-
 NAD+ mutant N191A, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 0.314
-
 NAD+ mutant N191A/N300A, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 0.4
-
 D-mannitol wild-type, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 0.9
-
 D-mannitol mutant N191L, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 1.1
-
 D-fructose mutant N191L, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 8.7
-
 D-mannitol mutant N191A, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 9
-
 D-fructose mutant N191A, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 20
-
 D-fructose mutant N191A/N300A, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 1187
-
 D-mannitol mutant N191A/N300A, pH 10.0, 25°C Pseudomonas fluorescens

Organism

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

Reaction

EC Number Reaction Comment Organism Reaction ID
1.1.1.67 D-mannitol + NAD+ = D-fructose + NADH + H+ the oxyanion hole of mannitol 2-dehydrogenase drives a precatalytic conformational equilibrium at the ternary complex level in which the reactive group of the substrate is activated for chemical conversion through its precise alignment with the unprotonated side chain of Lys295 in mannitol oxidation and C=O bond polarization by the carboxamide moieties of Asn191 and Asn300 in fructose reduction. In the subsequent hydride transfer step, the two asparagine residues provide about 40 kJ/mol of electrostatic stabilization Pseudomonas fluorescens
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.67 D-fructose + NADH + H+
-
 Pseudomonas fluorescens D-mannitol + NAD+
-
 ?
1.1.1.67 D-mannitol + NAD+
-
 Pseudomonas fluorescens D-fructose + NADH + H+
-
 ?

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.67 0.00045
-
 NADH mutant N191A/N300A, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 0.04
-
 NAD+ mutant N191A/N300A, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 0.55
-
 NAD+ mutant N191L, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 0.55
-
 NADH mutant N191L, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 0.56
-
 NADH mutant N191A, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 2.78
-
 NAD+ mutant N191A, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 40
-
 NAD+ wild-type, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 61
-
 NADH wild-type, pH 7.1, 25°C Pseudomonas fluorescens

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.67 0.000026
-
 D-fructose mutant N191A/N300A, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 0.000034
-
 D-mannitol mutant N191A/N300A, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 0.019
-
 NADH mutant N191A/N300A, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 0.064
-
 D-fructose mutant N191A, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 0.127
-
 NAD+ mutant N191A/N300A, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 0.319
-
 D-mannitol mutant N191A, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 0.407
-
 D-fructose mutant N191L, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 0.598
-
 D-mannitol mutant N191L, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 8.968
-
 NAD+ mutant N191A, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 9.964
-
 NAD+ mutant N191L, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 32
-
 NADH mutant N191A, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 100
-
 D-mannitol wild-type, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 170
-
 NADH mutant N191L, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 250
-
 D-fructose wild-type, pH 7.1, 25°C Pseudomonas fluorescens
1.1.1.67 400
-
 NAD+ wild-type, pH 10.0, 25°C Pseudomonas fluorescens
1.1.1.67 910
-
 NADH wild-type, pH 7.1, 25°C Pseudomonas fluorescens