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Information on EC 1.1.1.67 - mannitol 2-dehydrogenase
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1.1.1.67 mannitol 2-dehydrogenaseSpecify your search results
Word Map
- 1.1.1.67
-
polyol
- fluorescens
- d-fructose
-
1.1.1.138
-
5-dehydrogenase
-
heterofermentative
- reuteri
- molasses
- arabitol
- gluconobacter
- pfldh
- industry
- nutrition
- biotechnology
- synthesis
- analysis
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Synonyms
D-mannitol dehydrogenase, M2DH, mannitol 2-dehydrogenase, mannitol dehydrogenase, mannitol-2-dehydrogenase, MDH, mt-dh, MtDH, MtlD, NAD+-dependent mannitol dehydrogenase, 
more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
D-mannitol dehydrogenase
M2DH
mannitol 2-dehydrogenase
mannitol dehydrogenase
mannitol-2-dehydrogenase
MDH
MtDH
MtlD
NAD+-dependent mannitol dehydrogenase
polyol dehydrogenase
TM0298
D-mannitol dehydrogenase
-
-
-
-
mannitol dehydrogenase
-
-
-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
D-mannitol + NAD+ = D-fructose + NADH + H+
-
-
-
-
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
D-mannitol + NAD+ = D-fructose + NADH + H+
binding of substrate and NAD(H) is random for both D-mannitol oxidation and D-fructose reduction. Hydride transfer is rate-determining for D-fructose reduction. Product release steps control the maximum rates in the other direction of the enzymatic reaction
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
redox reaction
-
-
-
-
reduction
oxidation
-
-
-
-
reduction
-
-
-
-
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SYSTEMATIC NAME
IUBMB Comments
D-mannitol:NAD+ 2-oxidoreductase
-
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CAS REGISTRY NUMBER
COMMENTARY
9001-65-4
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
D-fructose + polyethylenimine-NH-succinyl-NADH + H+
D-mannitol + polyethylenimine-NH-succinyl-NAD+
-
-
-
-
r
D-glucitol + NAD+
?
4% of the activity with D-mannitol
-
-
?
D-mannitol + polyethyleneglycol-NH-succinyl-aminoethyl-NAD+
D-fructose + polyethyleneglycol-NH-succinyl-aminoethyl-NADH
-
-
-
?
D-mannitol + polyethyleneglycol-NH-succinyl-NAD+
D-fructose + polyethyleneglycol-NH-succinyl-NADH
-
-
-
?
D-mannitol + polyethylenimin-NH-succinyl-NAD+
D-fructose + polyethylenimin-NH-succinyl-NADH
-
-
-
?
D-tagatose + NAD(P)+
? + NAD(P)H
29% relative activity compared to D-fructose
-
-
?
D-tagatose + NAD+
?
29% relative activity on D-tagatose compared to 100% activity on D-fructose
-
-
?
D-xylulose + NAD(P)+
? + NAD(P)H
18% relative activity compared to D-fructose
-
-
?
D-xylulose + NAD+
?
18% relative activity on D-xylulose compared to 100% activity on D-fructose
-
-
?
L-sorbose + NAD(P)+
? + NAD(P)H
5% relative activity compared to D-fructose
-
-
?
D-fructose + NADH + H+
D-mannitol + NAD+
-
-
-
r
D-fructose + NADH + H+
D-mannitol + NAD+
-mannitol production
-
-
r
D-fructose + NADH + H+
D-mannitol + NAD+
-
-
-
r
D-fructose + NADH + H+
D-mannitol + NAD+
-mannitol production
-
-
r
D-fructose + NADH + H+
D-mannitol + NAD+
-
Asn300 has an auxiliary role in stabilization of the transition state of hydride transfer and His303 contributes to substrate positioning, role of Lys295 in general base enzymic catalysis
-
-
?
D-fructose + NADPH + H+
D-mannitol + NADP+
also active on fructose with NADPH
-
-
?
D-fructose + NADPH + H+
D-mannitol + NADP+
Thermotoga maritima MSB8 / DSM 3109 / ATCC 43589
also active on fructose with NADPH
-
-
?
D-mannitol + NAD(P)+
D-fructose + NAD(P)H + H+
Thermotoga maritima MSB8 / DSM 3109 / ATCC 43589
-
-
-
r
D-mannitol + NAD+
D-fructose + NADH + H+
free energy profiles for the enzymatic reaction suggest that enzyme primarily acts in D-mannitol oxidation
-
-
r
D-mannitol + NAD+
D-fructose + NADH + H+
-
enzyme highly specific for D-mannitol and D-fructose
-
r
D-mannitol + NAD+
D-fructose + NADH + H+
-
enzyme highly specific for D-mannitol and D-fructose
-
r
D-mannitol + NAD+
D-fructose + NADH + H+
-
the epsilon-NH2 group of Lys295 participates in an obligatory pH-dependent, pre-catalytic equilibrium which may control alcohol/alkoxide equilibration of the enzyme-bound D-mannitol and activates the C2 atom for subsequent catalytic oxidation by NAD+
-
-
?
D-mannitol + NAD+
D-fructose + NADH + H+
wild-type enzyme
-
-
r
D-mannitol + NAD+
D-fructose + NADH + H+
Thermotoga maritima MSB8 / DSM 3109 / ATCC 43589
100% activity
-
-
r
D-mannitol + NADP+
D-fructose + NADPH + H+
M2DH is a much poorer enzyme when it employes NADP+ and NADPH as compared to NAD+ and NADH
-
-
r
D-mannitol + NADP+
D-fructose + NADPH + H+
32% of the activity with NAD+
-
-
?
L-sorbose + NADH + H+
L-sorbitol + NAD+
5% relative activity on L-sorbose compared to 100% activity on D-fructose
-
-
?
additional information
?
-
a D-arabo configuration is required for a polyol substrate to become reactive. The C2 (R) configuration as in mannitol is preferred over the C2 (S) configuration as in D-sorbitol
-
-
?
additional information
?
-
-
slightly active with sorbitol, no activity with ribitol, arabinitol, or mesoerythritol
-
-
?
additional information
?
-
-
among the Escherichia coli strains, BL21 (DE3) plysS exhibits the maximum expression level of MDH (11mg/L)
-
-
?
additional information
?
-
no activity with glycerol, 1, 2-hexanediol, and 1,2,3-hexanetriol
-
-
?
additional information
?
-
-
no activity with glycerol, 1, 2-hexanediol, and 1,2,3-hexanetriol
-
-
?
additional information
?
-
-
no activity with NADP+ and NADPH
-
-
?
additional information
?
-
mannitol can be produced directly from glucose in a two-step enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60°C. No activity with glucose, xylose, threonine, arabinose, acetaldehyde, 2-butanone, sorbitol, xylitol, ethanol, or 2-butanol
-
-
?
additional information
?
-
-
mannitol can be produced directly from glucose in a two-step enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60°C. No activity with glucose, xylose, threonine, arabinose, acetaldehyde, 2-butanone, sorbitol, xylitol, ethanol, or 2-butanol
-
-
?
additional information
?
-
TM0298 shows no detectable activity on glucose, xylose, threonine, arabinose, acetaldehyde, 2-butanone, sorbitol, xylitol, ethanol, or 2-butanol
-
-
?
additional information
?
-
-
TM0298 shows no detectable activity on glucose, xylose, threonine, arabinose, acetaldehyde, 2-butanone, sorbitol, xylitol, ethanol, or 2-butanol
-
-
?
additional information
?
-
Thermotoga maritima MSB8 / DSM 3109 / ATCC 43589
mannitol can be produced directly from glucose in a two-step enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60°C. No activity with glucose, xylose, threonine, arabinose, acetaldehyde, 2-butanone, sorbitol, xylitol, ethanol, or 2-butanol
-
-
?
additional information
?
-
Thermotoga maritima MSB8 / DSM 3109 / ATCC 43589
TM0298 shows no detectable activity on glucose, xylose, threonine, arabinose, acetaldehyde, 2-butanone, sorbitol, xylitol, ethanol, or 2-butanol
-
-
?
additional information
?
-
-
the enzyme shows no activity with xylitol, inositol, sorbitol, rhamnose, mannose and xylose, and with NADPH and NADP+ as cofactors
-
-
?
additional information
?
-
-
the enzyme shows no activity with xylitol, inositol, sorbitol, rhamnose, mannose and xylose, and with NADPH and NADP+ as cofactors
-
-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
D-fructose + NADH + H+
D-mannitol + NAD+
-mannitol production
-
-
r
D-fructose + NADH + H+
D-mannitol + NAD+
-mannitol production
-
-
r
D-mannitol + NAD+
D-fructose + NADH + H+
free energy profiles for the enzymatic reaction suggest that enzyme primarily acts in D-mannitol oxidation
-
-
r
D-mannitol + NAD+
D-fructose + NADH + H+
-
-
-
-
?
D-mannitol + NAD+
D-fructose + NADH + H+
-
-
-
-
?
D-mannitol + NAD+
D-fructose + NADH + H+
wild-type enzyme
-
-
r
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NAD(P)+
its catalytic efficiency is 33times higher with NAD+ than with NADP+
NAD(P)H
MtDH has a higher Vmax with NADPH than with NADH, whereas its catalytic efficiency is 2.2times higher with NADH than with NADPH. Cofactor specificity is due to the high density of negatively charged residues (Glu193, Asp195, and Glu196) downstream of the NAD(P) interaction site, the glycine motif
additional information
only trace activity for utilization of NADP+
-
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
NAD+
although MtDH has a higher Vmax with NADPH than with NADH, its catalytic efficiency is 33 times higher with NAD+ than with NADP+
NAD+
dependent on, M2DH is a much poorer enzyme when it employes NADP+ and NADPH as compared to NAD+ and NADH
NADH
although MtDH has a higher Vmax with NADPH than with NADH, its catalytic efficiency is 2.2times higher with NADH than with NADPH
NADP+
400fold preference of the enzyme for NAD+ as compared to NADP+
NADP+
M2DH is a much poorer enzyme when it employes NADP+ and NADPH as compared to NAD+ and NADH
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Co2+
Zn2+
additional information
additional information
20 mM MgCl2 or CaCl2 do not increase the activity of the EDTA-treated enzyme
additional information
-
20 mM MgCl2 or CaCl2 do not increase the activity of the EDTA-treated enzyme
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
CuSO4
-
1 mM, 28% inhibition of D-fructose reduction, 18% inhibition of D-sorbitol oxidation
EDTA
20 mM, complete inhibition, can be restored by 20 mM Zn2+, but not by Mn2+ or Mg2+
EDTA
activity of MtDH pretreated with 10 mM EDTA for 20 min at 37°C decreases 96%. Adding 20 mM ZnCl2 to the EDTA-treated enzyme restores activity up to 80% of the control (enzyme not treated with EDTA). Adding 20 mM CoCl2 and 20 mM MnCl2 to the EDTA-treated enzyme restores activity up to 132% and 94% of the control, respectively. In contrast, adding 20 mM MgCl2 or CaCl2 does not increase the activity of the EDTA-treated enzyme.; activity of MtDH pretreated with 10 mM for 20 min at 37°C decreases 96%. Adding 20 mM ZnCl2 to the EDTA-treated enzyme restores activity up to 80% of the control. Adding 20 mM CoCl2 and 20 mM MnCl2 to the EDTA-treated enzyme restores activity up to 132% and 94% of the control, respectively
NaCl
the enzyme displays a 4.37fold decrease of activity in 600 mM and about 1000fold decrease of activity in 1000 mM NaCl
NaCl
-
with increasing NaCl concentrations enzyme activity decreases, 1.5 M NaCl 74% inhibition for oxidation and 72% inhibition for reduction
additional information
-
no inhibitory effect on D-fructose reduction detected for MgSO4, CaSO4, Na2SO4, ZnSO4 and MnSO4
-
additional information
no inhibition of M2DH by bovine serum albumin
-
additional information
-
no inhibition of M2DH by bovine serum albumin
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
no clear stimulatory effect of MgSO4+, MnSO4, CuSO4, ZnSO4, Na2SO4 and CaSO4 on activity
-
additional information
no activation of M2DH by bovine serum albumin
-
additional information
-
no activation of M2DH by bovine serum albumin
-
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DISEASE
TITLE OF PUBLICATION
LINK TO PUBMED
Malaria
Analysis of quaternary structure of a [LDH-like] malate dehydrogenase of Plasmodium falciparum with oligomeric mutants.
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
33.15
D-arabitol
mutant enzyme H303A/R73A/K381A, at pH 7.4 and 25°C
0.24
D-fructose
wild-type, pH 7.1, temperature not specified in the publication
0.54
D-fructose
mutant N191D, pH 10.0, temperature not specified in the publication
3.9
D-fructose
mutant N300D, pH 10.0, temperature not specified in the publication
6
D-fructose
mutant N191D, pH 7.1, temperature not specified in the publication
20
D-fructose
mutant N191D/N300D, pH 10.0, temperature not specified in the publication
22
D-fructose
mutant N300D, pH 6.8, temperature not specified in the publication
0.3
D-mannitol
mutant N300D, pH 10.0, temperature not specified in the publication
0.32
D-mannitol
mutant N191D, pH 10.0, temperature not specified in the publication
8.15
D-mannitol
mutant enzyme H303A/R73A/K381A, at pH 7.4 and 25°C
9
D-mannitol
mutant N191D, pH 7.1, temperature not specified in the publication
21
D-mannitol
wild-type, pH 7.1, temperature not specified in the publication
93
D-mannitol
mutant N300D, pH 6.8, temperature not specified in the publication
1800
meso-erythritol
Km far above 1800 mM, mutant enzyme E133A, at pH 7.4 and 25°C
1800
meso-erythritol
Km far above 1800 mM, mutant enzyme E133Q, at pH 7.4 and 25°C
1800
meso-erythritol
Km far above 1800 mM, mutant enzyme H303A, at pH 7.4 and 25°C
1800
meso-erythritol
Km far above 1800 mM, mutant enzyme N191A, at pH 7.4 and 25°C
1800
meso-erythritol
Km far above 1800 mM, mutant enzyme N300A, at pH 7.4 and 25°C
1800
meso-erythritol
Km far above 1800 mM, mutant enzyme N300S, at pH 7.4 and 25°C
1800
meso-erythritol
Km far above 1800 mM, wild type enzyme, at pH 7.4 and 25°C
0.001
NAD+
mutant N300D, pH 6.8, temperature not specified in the publication
0.054
NAD+
mutant N191D, pH 10.0, temperature not specified in the publication
0.074
NAD+
mutant N300D, pH 10.0, temperature not specified in the publication
0.231
NAD+
mutant N191D, pH 7.1, temperature not specified in the publication
0.775
NAD+
wild-type, pH 7.1, temperature not specified in the publication
0.008
NADH
mutant N191D, pH 7.1, temperature not specified in the publication
0.009
NADH
mutant N300D, pH 10.0, temperature not specified in the publication
0.011
NADH
mutant N191D/N300D, pH 10.0, temperature not specified in the publication
0.016
NADH
mutant N191D, pH 10.0, temperature not specified in the publication
0.02
NADH
mutant N300D, pH 6.8, temperature not specified in the publication
0.067
NADH
wild-type, pH 7.1, temperature not specified in the publication
additional information
additional information
steady-state kinetic analysis, recombinant wild-type and mutant enzymes, overview
-
additional information
additional information
-
steady-state kinetic analysis, recombinant wild-type and mutant enzymes, overview
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0017
D-arabitol
mutant enzyme H303A/R73A/K381A, at pH 7.4 and 25°C
0.15
D-fructose
mutant N191D/N300D, pH 10.0, temperature not specified in the publication
0.8
D-fructose
mutant N300D, pH 10.0, temperature not specified in the publication
0.9
D-fructose
mutant N300D, pH 6.8, temperature not specified in the publication
2.7
D-fructose
mutant N191D, pH 7.1, temperature not specified in the publication
8.2
D-fructose
mutant N191D, pH 10.0, temperature not specified in the publication
61
D-fructose
wild-type, pH 7.1, temperature not specified in the publication
0.00055
D-mannitol
mutant N191D, pH 7.1, temperature not specified in the publication
0.001
D-mannitol
mutant enzyme H303A/R73A/K381A, at pH 7.4 and 25°C
0.0011
D-mannitol
mutant N191D, pH 10.0, temperature not specified in the publication
0.0014
D-mannitol
mutant N300D, pH 6.8, temperature not specified in the publication
0.0015
D-mannitol
mutant N300D, pH 10.0, temperature not specified in the publication
15.9
D-mannitol
wild-type, pH 7.1, temperature not specified in the publication
0.0012
meso-erythritol
mutant enzyme N191A, at pH 7.4 and 25°C
0.0024
meso-erythritol
mutant enzyme N300S, at pH 7.4 and 25°C
0.0036
meso-erythritol
mutant enzyme N300A, at pH 7.4 and 25°C
0.0159
meso-erythritol
mutant enzyme H303A, at pH 7.4 and 25°C
0.489
meso-erythritol
mutant enzyme E133Q, at pH 7.4 and 25°C
0.538
meso-erythritol
mutant enzyme E133A, at pH 7.4 and 25°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.000051
D-arabitol
mutant enzyme H303A/R73A/K381A, at pH 7.4 and 25°C
0.0074
D-fructose
mutant N191D/N300D, pH 10.0, temperature not specified in the publication
0.041
D-fructose
mutant N300D, pH 6.8, temperature not specified in the publication
0.205
D-fructose
mutant N300D, pH 10.0, temperature not specified in the publication
0.45
D-fructose
mutant N191D, pH 7.1, temperature not specified in the publication
15
D-fructose
mutant N191D, pH 10.0, temperature not specified in the publication
250
D-fructose
wild-type, pH 7.1, temperature not specified in the publication
0.000015
D-mannitol
mutant N300D, pH 6.8, temperature not specified in the publication
0.000061
D-mannitol
mutant N191D, pH 7.1, temperature not specified in the publication
0.00016
D-mannitol
mutant enzyme H303A/R73A/K381A, at pH 7.4 and 25°C
0.0034
D-mannitol
mutant N191D, pH 10.0, temperature not specified in the publication
0.0049
D-mannitol
mutant N300D, pH 10.0, temperature not specified in the publication
0.757
D-mannitol
wild-type, pH 7.1, temperature not specified in the publication
0.0000021
meso-erythritol
mutant enzyme N191A, at pH 7.4 and 25°C
0.0000044
meso-erythritol
mutant enzyme N300S, at pH 7.4 and 25°C
0.0000065
meso-erythritol
mutant enzyme N300A, at pH 7.4 and 25°C
0.000029
meso-erythritol
mutant enzyme H303A, at pH 7.4 and 25°C
0.00088
meso-erythritol
mutant enzyme E133Q, at pH 7.4 and 25°C
0.00097
meso-erythritol
mutant enzyme E133A, at pH 7.4 and 25°C
0.0024
NAD+
mutant N191D, pH 7.1, temperature not specified in the publication
0.02
NAD+
mutant N191D, pH 10.0, temperature not specified in the publication
0.02
NAD+
mutant N300D, pH 10.0, temperature not specified in the publication
1.4
NAD+
mutant N300D, pH 6.8, temperature not specified in the publication
20
NAD+
wild-type, pH 7.1, temperature not specified in the publication
15
NADH
mutant N191D/N300D, pH 10.0, temperature not specified in the publication
45
NADH
mutant N300D, pH 6.8, temperature not specified in the publication
89
NADH
mutant N300D, pH 10.0, temperature not specified in the publication
340
NADH
mutant N191D, pH 7.1, temperature not specified in the publication
510
NADH
mutant N191D, pH 10.0, temperature not specified in the publication
910
NADH
wild-type, pH 7.1, temperature not specified in the publication
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.003
NAD+
mutant N191D, pH 7.1, temperature not specified in the publication
0.07
NAD+
mutant N300D, pH 6.8, temperature not specified in the publication
0.08
NAD+
wild-type, pH 7.1, temperature not specified in the publication
0.061
NADH
mutant N300D, pH 6.8, temperature not specified in the publication
0.064
NADH
mutant N191D, pH 7.1, temperature not specified in the publication
0.08
NADH
wild-type, pH 7.1, temperature not specified in the publication
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
14
crude extract, at 25°C, using 100 mM Tris-HCl buffer, pH 7.1
33
after 6.7fold purification, at 25°C, using 100 mM Tris-HCl buffer, pH 7.1
54
63
-
MDH expressed in Escherichia coli strain BL21 (DE3) plysS, with fructose as substrate
additional information
-
the activity for inositol, sorbitol, galactitol, ribitol, xylitol, arabitol and erythritol is very low
54
purified enzyme at 80°C with D-fructose as the substrate and NADH as the cofactor
54
purified enzyme, at 80°C with fructose as the substrate and NADH as the cofactor
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
10
5.3
5.5
5.5 - 6
6 - 7.2
6.5
8.6
8.9 - 10
additional information
5.5 - 6
-
optimal pH in den range of 5.5-6.0 for reduction of fructose
5.5 - 6
optimally reduces L-fructose at pH values between pH 5.5 and 6.0
additional information
pH 5.5-6 is the optimum for D-fructose reduction and pH 8.3-8.6 is the optimum for mannitol reduction
additional information
-
pH 5.5-6 is the optimum for D-fructose reduction and pH 8.3-8.6 is the optimum for mannitol reduction
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.5 - 7
-
pH 4.5: about 80% of maximal activity, pH 7.0: about 60% of maximal activity, reduction of D-fructose
5.5 - 9.5
about 80% activity at pH 5.5, 100% activity at pH 6.5, about 90% activity at pH 7.5, about 85% activity at pH 8.5, about 70% activity at pH 9.5
6 - 6.5
6 - 9
-
more than 60% of the maximal activity in the pH range 6.0â9.0
6.8 - 8.3
-
pH 6.8: about 20% of maximal activity, pH 8.3: about 55% of maximal activity, oxidation of D-mannitol
9 - 9.5
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
35
40
50
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20 - 40
-
20°C: about 85% of maximal activity, 40°C: about 70% of maximal activity, reduction of D-fructose
20 - 50
about 40% activity at 20°C, about 50% activity at 25°C, about 70% activity at 30°C, 100% activity at 35°C, about 98% activity at 40°C, about 90% activity at 45°C, about 30% activity at 50°C
90 - 120
MtDH retains 63% of its activity at 120°C but shows no detectable activity at 25°C
95 - 120
retains 63% of its activity at 120°C but shows no detectable activity at room temperature
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain ATCC12291, gene mdh
SwissProt
brenda
-
-
-
brenda
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
-
glucose as carbon source represses mannitol dehydrogenase
brenda
[Brevibacterium] flavum 2247 / ATCC 14067
-
glucose as carbon source represses mannitol dehydrogenase
-
brenda
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
Show AA Sequence and Transmembrane Helices (977 entries)
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
132000 - 138000
-
gel filtration, sucrose density gradient centrifugation
36000
53000
54000
55000
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homotetramer
monomer
octamer
gel filtration at room temperature, MtDH in solution
tetramer
dimer
gel filtration at room temperature, MtDH in solution
tetramer
gel filtration at room temperature, MtDH in solution
tetramer
gel filtration or analytical ultracentrifugation, MtDH in solution
tetramer
Thermotoga maritima MSB8 / DSM 3109 / ATCC 43589
-
gel filtration or analytical ultracentrifugation, MtDH in solution
-
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, binary complex of selenomethionine substituted proteins with NAD(H) and a ternary complex with NAD(H) and D-mannitol have been determined to resolutions of 1.7 and 1.8 A respectively
-
hanging drop vapor diffusion method, binary complex with NAD+ and ternary complex with NAD+ and D-mannitol have been determined to resolutions of 1.7 and 1.8 A and R-factors of 0.171 and 0.176 respectively
-
purified recombinant His-tagged enzyme, 10 mg/ml protein in 50 mM Tris-HCl, pH 8.5, room temperature, microbatch-under-oil method, mixing of equal volumes of protein and crystallization solutions, the latter containing 30% 2-methyl-2,4-pentanediol, and 0.1 M Na HEPES, pH 7.5, X-ray diffraction structure determination and analysis at 3.3 A resolution, molecular-replacement
-
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D230A
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
D69A
E133A
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
E133Q
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
E292A
mutation partially disrupts the catalytic cycle. Role for residue Glu292 as a gate in a water chain mechanism of proton translocation. Removal of gatekeeper control in the E292A mutant results in a selective, up to 120fold slowing down of microscopicsteps immediately preceding catalytic oxidation of mannitol, consistent with the notion that formation of the productive enzyme-NAD-mannitol complex is promoted by a corresponding position change of Glu292
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)
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
H303A
H303A/R373A/K381A
the mutant shows severely reduced catalytic efficiency compared to the wild type enzyme
K295A
K295M
-
2000000fold lower turnover number for D-mannitol oxidation at pH 10.0 than the wild-type enzyme
K381A
the mutant shows severely reduced catalytic efficiency compared to the wild type enzyme
N191A
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
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
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
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 enzymeâNAD+-mannitol complex
N300A
N300D
N300S
the mutant shows severely reduced catalytic efficiency compared to the wild type enzyme
R373A
the mutant shows severely reduced catalytic efficiency compared to the wild type enzyme
additional information
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)
D69A
utilizes NAD(H) and NADP(H) with similar catalytic efficiencies. Uses NADP(H) almost as well as wild-type enzyme uses NAD(H)
H303A
-
mutant enzyme displays catalytic efficiency for NAD+-dependent oxidation of D-mannitol 300fold below the wild-type value
H303A
the mutant shows severely reduced catalytic efficiency compared to the wild type enzyme
K295A
-
30000fold lower turnover number for D-mannitol oxidation at pH 10.0 than the wild-type enzyme
K295A
-
mutant enzyme displays catalytic efficiency for NAD+-dependent oxidation of D-mannitol 400000fold below the wild-type value
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 enzymeâNAD+-mannitol complex
N191A
the mutant shows severely reduced catalytic efficiency compared to the wild type enzyme
N300A
-
mutant enzyme displays catalytic efficiency for NAD+-dependent oxidation of D-mannitol 1000fold below the wild-type value
N300A
the mutant shows severely reduced catalytic efficiency compared to the wild type enzyme
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
N300D
the mutant shows severely reduced catalytic efficiency compared to the wild type enzyme
additional information
D-mannitol production by resting state whole cell biotransformation of D-fructose by heterologous mannitol dehydrogenase gene from Leuconostoc pseudomesenteroides and the formate dehydrogenase gene, gene fdh from Mycobacterium vaccae N10, expression in Bacillus megaterium, development of an in vivo system, overview
additional information
-
D-mannitol production by resting state whole cell biotransformation of D-fructose by heterologous mannitol dehydrogenase gene from Leuconostoc pseudomesenteroides and the formate dehydrogenase gene, gene fdh from Mycobacterium vaccae N10, expression in Bacillus megaterium, development of an in vivo system, overview
-
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5 - 9
6 - 6.5
6 - 8
8.5
TM0298 is sensitive to proteolytic degradation in purification protocols at pH 8.5
695814
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30 - 40
-
The enzyme is very stable at 30°C. The half life time at 40°C is 35 min for D-mannitol oxidation.
40
75 - 90
-
the enzyme records 85%, 50% and 10% of its initial activity after 4 h of exposure to 75, 80, 85 and 90°C, respectively. The enzyme almost does not lose the activity after 1 h of incubation at 75°C and still has half of initial activity after incubated at 90°C for 1 h
80
95
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-16°C, 0.02 M sodium acetate buffer pH 6.0, 1 mM 2-mercaptoethanol, several weeks
-
4°C or 20°C, enzyme loses approximately 60% of its specific activity after storage for 3 weeks in presence of 1 mM DTT, without DTT the same loss is observed after 2 days
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Co2+ chelating Sepharose column chromatography and Superdex 200 gel filtration
histidine-tagged recombinant protein, expressed in Escherichia coli
-
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
-
recombinant protein
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli
expression in Escherichia coli, effecting strong catalytic activity of an NADH-dependent reduction of D-fructose to D-mannitol in cell extracts of the recombinant Escherichia coli strain
expression of wild-type and mutant enzymes in Escherichia coli strain JM109
expression of wild-type enzyme and mutant enzymes in Escherichia coli
-
fusion of six His codons to the 3'-end of the mdh gene and expression in Escherichia coli M15. The enzyme shares significant sequence similarity with the medium-chain dehydrogenase/reductase protein family
gene mdh, high level expression in Bacillus megaterium requiring the adaptation of the corresponding ribosome binding site, the fdh gene is adapted to Bacillus megaterium codon usage via complete chemical gene synthesis, overview
gene mtdh, expression of the His-tagged enzyme in Escherichia coli strain BL21(DE3)
-
subcloned into vector pDEST110 and overexpressed in different strains of Escherichia coli (BL21 (DE3) plysS, JM109, Origami(DE3) or M15)
-
the TM0298 gene subcloned into the NdeI and XhoI sites of pET24a(+) to yield plasmid pTmMtDH, from which MtDH is expressed with a C-terminal His6-tag in Escherichia coli BL21(DE3)
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
-
sensitive and specific photometric determination of mannitol in human serum
biotechnology
recombinant Escherichia coli expressing the enzyme from Leuconostoc pseudomesenteroides expressing strong catalytic activity of an NADH-dependent reduction of D-fructose to D-mannitol in cell extracts of the recombinant Escherichia coli strain can be utilized as an efficient biocatalyst for D-mannitol formation
industry
nutrition
synthesis
industry
-
best strain for expression of MDH in both laboratory and industrial applications is Escherichia coli BL21 (DE3) plysS
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
synthesis
the recombinant enzyme expressed in Bacillus megaterium is useful in production of D-mannitol using a resting cell biotransformation approach
synthesis
-
the recombinant enzyme expressed in Bacillus megaterium is useful in production of D-mannitol using a resting cell biotransformation approach
-
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Hult, K.; Veide, A.; Gatenbeck, S.
The distribution of the NADPH regenerating mannitol cycle among fungal species
Arch. Microbiol.
128
253-255
1980
Fomes pinicola
brenda
Schmatz D.M.; Baginsky W.F.; Turner, M.J.
Evidence for and characterization of a mannitol cycle in Eimeria tenella
Mol. Biochem. Parasitol.
32
263-270
1989
Eimeria tenella
brenda
Sasajima, K.; Isono, M.
Polyol dehydrogenases in the soluble fraction of Acetobacter melanogenum
Agric. Biol. Chem.
32
161-169
1968
Gluconobacter oxydans
-
brenda
Schneider, K.H.; Giffhorn, F.
Purification and properties of a polyol dehydrogenase from the phototrophic bacterium Rhodobacter sphaeroides
Eur. J. Biochem.
184
15-19
1989
Luteovulum sphaeroides
brenda
Quain, D.E.; Boulton, C.A.
Growth and metabolism of mannitol by strains of Saccharomyces cerevisiae
J. Gen. Microbiol.
133
1675-1684
1987
Saccharomyces cerevisiae
brenda
Richter, D.F.E.; Kirst, G.O.
D-Mannitol dehydrogenase and D-mannitol-1-phosphate dehydrogenase in Platymonas subcordiformis: some characteristics and their role in osmotic adaptation
Planta
170
528-534
1987
Tetraselmis subcordiformis
brenda
Mori, M.; Shiio, I.
Pyruvate formation and sugar metabolism in an amino acid-producing bacterium, Brevibacterium flavum
Agric. Biol. Chem.
51
129-138
1987
[Brevibacterium] flavum, [Brevibacterium] flavum 2247 / ATCC 14067
-
brenda
Kulbe, K.D.; Schwab, U.; Howaldt, M.; Kimmerle, K.
Anwendung von Membranverfahren bei der simultanen Herstellung von Mannitol und Gluconsäure aus Saccharose durch konjugierte NAD+-abhängige Dehydrogenasen
GBF Monogr.
9
189-200
1986
Saccharomyces cerevisiae
-
brenda
Mathis, J.N.; Barbour, W.M.; Miller, T.B.; Israel, D.W.; Elkan, G.H.
Characterization of a mannitol-utilizing, nitrogen-fixing Bradyrhizobium japonicum USDA 110 derivative
Appl. Environ. Microbiol.
52
81-85
1986
Bradyrhizobium japonicum
brenda
Davis, C.L.; Robb, F.T.
Maintenance of different mannitol uptake systems during starvation in oxidative and fermentative marine bacteria
Appl. Environ. Microbiol.
50
743-748
1985
Pseudomonas sp.
brenda
Allenza, P.; Lee, Y.N.; Lessie, T.G.
Enzymes related to fructose utilization in Pseudomonas cepacia
J. Bacteriol.
150
1348-1356
1982
Burkholderia cepacia
brenda
Mulongoy, K.; Elkan, G.H.
Some effects of mannitol on the glucose metabolism of two derivatives of a strain of Rhizobium japonicum differing in mannitol dehydrogenase
Curr. Microbiol.
1
335-340
1978
Bradyrhizobium japonicum
-
brenda
Yamanaka, K.; Izawa, K.; Tenmizu, K.
Physicochemical properties of crystalline D-mannitol dehydrogenase of Leuconostoc mesenteroides
Agric. Biol. Chem.
41
1695-1699
1977
Leuconostoc mesenteroides
-
brenda
Mehta, R.J.; Fare, L.R.; Shearer, M.E.; Nash, C.H.
Mannitol oxidation in two Micromonospora isolates and in representative species of other actinomycetes
Appl. Environ. Microbiol.
33
1013-1015
1977
Gluconobacter oxydans, Streptomyces lavendulae, Actinoplanes missouriensis, Levilactobacillus brevis, Mycolicibacterium smegmatis, Nocardia erythropolis, Sinorhizobium meliloti
brenda
Kuykendall, L.D.; Elkan, G.H.
Some features of mannitol metabolism in Rhizobium japonicum
J. Gen. Microbiol.
98
291-295
1977
Bradyrhizobium japonicum
brenda
Ueng, S.T.H.; McGuinness, E.T.
D-Mannitol dehydrogenase from Absidia glauca. Steady-state kinetic properties and the inhibitory role of mannitol 1-phosphate
Biochemistry
16
107-111
1977
Absidia glauca
brenda
Ueng, S.T.H.; Hartanowicz, P.; Lewandowski, C.; Keller, J.; M.Holick, E.T.McGuinness
D-Mannitol dehydrogenase from Absidia glauca. Purification, metabolic role, and subunit interactions
Biochemistry
15
1743-1749
1976
Absidia glauca
brenda
Yamanaka, K.
D-Mannitol dehydrogenase from Leuconostoc mesenteroides
Methods Enzymol.
41B
138-142
1975
Leuconostoc mesenteroides, Levilactobacillus brevis, Lactobacillus gayonii, Lactobacillus pentoaceticus
brenda