Literature summary for 1.1.1.67 extracted from

Klimacek, M.; Nidetzky, B.
From alcohol dehydrogenase to a "one-way" carbonyl reductase by active-site redesign: a mechanistic study of mannitol 2-dehydrogenase from Pseudomonas fluorescens (2010), J. Biol. Chem., 285, 30644-30653.

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Pseudomonas fluorescens

Protein Variants

Protein Variants	Comment	Organism
N191D	the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol is altered 10000- to 100000fold from being balanced in the wild-type enzyme to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 0.1 and 6.3 0.2, respectively	Pseudomonas fluorescens
N191D/N300D	mutant behaves as a slow fructose reductase at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8-10	Pseudomonas fluorescens
N300D	the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol is altered 10000- to 100000fold from being balanced in the wild-type enzyme to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 0.1 and 6.3 0.2, respectively	Pseudomonas fluorescens

Inhibitors

Inhibitors	Comment	Organism	Structure
NAD+	-	Pseudomonas fluorescens
NADH	-	Pseudomonas fluorescens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.001	-	NAD+	mutant N300D, pH 6.8, temperature not specified in the publication	Pseudomonas fluorescens
0.008	-	NADH	mutant N191D, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
0.009	-	NADH	mutant N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.011	-	NADH	mutant N191D/N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.016	-	NADH	mutant N191D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.02	-	NADH	mutant N300D, pH 6.8, temperature not specified in the publication	Pseudomonas fluorescens
0.054	-	NAD+	mutant N191D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.067	-	NADH	wild-type, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
0.074	-	NAD+	mutant N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.231	-	NAD+	mutant N191D, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
0.24	-	D-fructose	wild-type, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
0.3	-	D-mannitol	mutant N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.32	-	D-mannitol	mutant N191D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.54	-	D-fructose	mutant N191D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.775	-	NAD+	wild-type, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
3.9	-	D-fructose	mutant N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
6	-	D-fructose	mutant N191D, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
9	-	D-mannitol	mutant N191D, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
20	-	D-fructose	mutant N191D/N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
21	-	D-mannitol	wild-type, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
22	-	D-fructose	mutant N300D, pH 6.8, temperature not specified in the publication	Pseudomonas fluorescens
93	-	D-mannitol	mutant N300D, pH 6.8, temperature not specified in the publication	Pseudomonas fluorescens

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
55000	-	x * 55000, SDS-PAGE	Pseudomonas fluorescens

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas fluorescens	O08355	-	-

Substrates and Products (Substrate)

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

Subunits

Subunits	Comment	Organism
?	x * 55000, SDS-PAGE	Pseudomonas fluorescens

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.00055	-	D-mannitol	mutant N191D, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
0.0011	-	D-mannitol	mutant N191D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.0014	-	D-mannitol	mutant N300D, pH 6.8, temperature not specified in the publication	Pseudomonas fluorescens
0.0015	-	D-mannitol	mutant N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.15	-	D-fructose	mutant N191D/N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.8	-	D-fructose	mutant N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.9	-	D-fructose	mutant N300D, pH 6.8, temperature not specified in the publication	Pseudomonas fluorescens
2.7	-	D-fructose	mutant N191D, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
8.2	-	D-fructose	mutant N191D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
15.9	-	D-mannitol	wild-type, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
61	-	D-fructose	wild-type, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens

Cofactor

Cofactor	Comment	Organism	Structure
NAD+	-	Pseudomonas fluorescens
NADH	-	Pseudomonas fluorescens

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism
0.003	-	NAD+	mutant N191D, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
0.061	-	NADH	mutant N300D, pH 6.8, temperature not specified in the publication	Pseudomonas fluorescens
0.064	-	NADH	mutant N191D, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
0.07	-	NAD+	mutant N300D, pH 6.8, temperature not specified in the publication	Pseudomonas fluorescens
0.08	-	NAD+	wild-type, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
0.08	-	NADH	wild-type, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.000015	-	D-mannitol	mutant N300D, pH 6.8, temperature not specified in the publication	Pseudomonas fluorescens
0.000061	-	D-mannitol	mutant N191D, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
0.0024	-	NAD+	mutant N191D, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
0.0034	-	D-mannitol	mutant N191D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.0049	-	D-mannitol	mutant N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.0074	-	D-fructose	mutant N191D/N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.02	-	NAD+	mutant N191D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.02	-	NAD+	mutant N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.041	-	D-fructose	mutant N300D, pH 6.8, temperature not specified in the publication	Pseudomonas fluorescens
0.205	-	D-fructose	mutant N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
0.45	-	D-fructose	mutant N191D, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
0.757	-	D-mannitol	wild-type, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
1.4	-	NAD+	mutant N300D, pH 6.8, temperature not specified in the publication	Pseudomonas fluorescens
15	-	D-fructose	mutant N191D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
15	-	NADH	mutant N191D/N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
20	-	NAD+	wild-type, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
45	-	NADH	mutant N300D, pH 6.8, temperature not specified in the publication	Pseudomonas fluorescens
89	-	NADH	mutant N300D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
250	-	D-fructose	wild-type, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
340	-	NADH	mutant N191D, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens
510	-	NADH	mutant N191D, pH 10.0, temperature not specified in the publication	Pseudomonas fluorescens
910	-	NADH	wild-type, pH 7.1, temperature not specified in the publication	Pseudomonas fluorescens