Information on EC 1.1.1.67 - mannitol 2-dehydrogenase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
1.1.1.67
-
RECOMMENDED NAME
GeneOntology No.
mannitol 2-dehydrogenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
D-mannitol + NAD+ = D-fructose + NADH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
redox reaction
-
-
-
-
reduction
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Fructose and mannose metabolism
-
-
mannitol cycle
-
-
SYSTEMATIC NAME
IUBMB Comments
D-mannitol:NAD+ 2-oxidoreductase
-
CAS REGISTRY NUMBER
COMMENTARY hide
9001-65-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain 2247 (ATCC14067)
-
-
Manually annotated by BRENDA team
strain 2247 (ATCC14067)
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Fomes pinicola
-
-
-
Manually annotated by BRENDA team
strain LMG 1489
-
-
Manually annotated by BRENDA team
Lactobacillus gayonii
-
-
-
Manually annotated by BRENDA team
Lactobacillus pentoaceticus
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
ATCC-9135
SwissProt
Manually annotated by BRENDA team
strain ATCC12291, gene mdh
SwissProt
Manually annotated by BRENDA team
no activity in Lactobacillus plantarum
-
-
-
Manually annotated by BRENDA team
Nocardia erythropolis
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
phototrophic bacterium
-
-
Manually annotated by BRENDA team
Sarcina aurantiaca
-
-
-
Manually annotated by BRENDA team
Sarcina marginata
-
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
arabitol + NAD(P)+
? + NAD(P)H
show the reaction diagram
-
-
-
-
?
D-arabinitol + NAD+
D-ribulose + NADH + H+
show the reaction diagram
D-arabinitol + NAD+
D-xylulose + NADH + H+
show the reaction diagram
-
-
-
-
r
D-arabitol + NAD+
? + NADH
show the reaction diagram
D-fructose + NAD(P)+
? + NAD(P)H
show the reaction diagram
-
-
-
-
?
D-fructose + NADH + H+
D-mannitol + NAD+
show the reaction diagram
D-fructose + NADPH + H+
D-mannitol + NADP+
show the reaction diagram
also active on fructose with NADPH
-
-
?
D-fructose 1-phosphate + NADH
?
show the reaction diagram
D-glucitol + NAD+
?
show the reaction diagram
-
4% of the activity with D-mannitol
-
-
?
D-glucitol + NAD+
? + NADH
show the reaction diagram
D-mannitol + NAD(P)+
D-fructose + NAD(P)H + H+
show the reaction diagram
D-mannitol + NAD+
D-fructose + NADH + H+
show the reaction diagram
D-mannitol + NADP+
D-fructose + NADPH + H+
show the reaction diagram
D-mannitol + polyethyleneglycol-NH-succinyl-aminoethyl-NAD+
D-fructose + polyethyleneglycol-NH-succinyl-aminoethyl-NADH
show the reaction diagram
-
-
-
-
D-mannitol + polyethyleneglycol-NH-succinyl-NAD+
D-fructose + polyethyleneglycol-NH-succinyl-NADH
show the reaction diagram
-
-
-
-
D-mannitol + polyethylenimin-NH-succinyl-NAD+
D-fructose + polyethylenimin-NH-succinyl-NADH
show the reaction diagram
-
-
-
-
D-tagatose + NAD(P)+
? + NAD(P)H
show the reaction diagram
29% relative activity compared to D-fructose
-
-
?
D-tagatose + NAD+
?
show the reaction diagram
29% relative activity on D-tagatose compared to 100% activity on D-fructose
-
-
?
D-xylulose + NAD(P)+
? + NAD(P)H
show the reaction diagram
18% relative activity compared to D-fructose
-
-
?
D-xylulose + NAD+
?
show the reaction diagram
18% relative activity on D-xylulose compared to 100% activity on D-fructose
-
-
?
D-xylulose + NADH + H+
D-arabinitol + NAD+
show the reaction diagram
-
-
-
-
?
isomaltulose + NAD(P)+
? + NAD(P)H
show the reaction diagram
-
-
-
-
?
L-sorbitol + NAD+
L-sorbose + NADH + H+
show the reaction diagram
L-sorbose + NAD(P)+
? + NAD(P)H
show the reaction diagram
5% relative activity compared to D-fructose
-
-
?
L-sorbose + NADH + H+
L-sorbitol + NAD+
show the reaction diagram
sorbitol + NAD(P)+
? + NAD(P)H
show the reaction diagram
-
-
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
D-fructose + NADH + H+
D-mannitol + NAD+
show the reaction diagram
D-mannitol + NAD+
D-fructose + NADH + H+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
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+
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CaCl2
-
10 mM, activates to 173% of control
KCl
-
10 mM, activates to 127% of control
LiCl
-
10 mM, activates to 126% of control
MgCl2
-
10 mM, activates to 149% of control
NH4Cl
-
10 mM, activates to 137% of control
NiCl2
-
10 mM, activates to 106% of control
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
citrate
-
50 mM, pH 5.4, 72% inhibition
Cu2+
-
1 mM, 61% inhibition
CuSO4
-
1 mM, 28% inhibition of D-fructose reduction, 18% inhibition of D-sorbitol oxidation
D-fructose
-
substrate inhibition, Ki: 1.356 mM
D-mannitol
-
product inhibition, Ki: 24.6 mM
FeCl2
-
10 mM, 88% inhibition
imidazole
-
175 mM,pH 7.0, 74% inhibition
L-sorbitol
-
Ki: 24.6 mM
mannitol 1-phosphate
-
-
Methyl mercurinitrate
-
complete inhibition
MnCl2
-
10 mM, 48% inhibition
NaCl
-
with increasing NaCl concentrations enzyme activity decreases, 1.5 M NaCl 74% inhibition for oxidation and 72% inhibition for reduction
p-chloromercuribenzoate
-
0.1 mM, 100% inhibition
p-hydroxymercuribenzoate
PCMB
0.1 mM, complete inhibition
Zn2+
-
1 mM, 94% inhibition
ZnCl2
-
10 mM, 60% inhibition
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.8 - 163
D-arabinitol
1.6 - 6.5
D-arabitol
0.24 - 79.2
D-fructose
32
D-glucitol
-
-
0.29 - 1187
D-mannitol
58.5
D-sorbitol
-
recombinant protein
1.7
D-xylulose
-
pH 7.1, 25C
680
L-sorbitol
-
pH 10.0, 25C
0.001 - 0.775
NAD+
0.0033 - 0.15
NADH
7.5
NADP+
at 80C and pH 8.3; at 80C, pH 8.3
0.17
NADPH
at 80C and pH 6.1; at 80C, pH 6.1
0.1
polyethyleneglycol-NH-succinyl-aminoethyl-NADH
-
-
-
0.125
polyethyleneglycol-NH-succinyl-NADH
-
-
-
0.074
polyethylenimine-NH-succinyl-NADH
-
-
-
additional information
additional information
-
steady-state kinetic analysis, recombinant wild-type and mutant enzymes, overview
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
162
D-arabinitol
Aspergillus fumigatus
-
pH 10.0, 25C
0.15 - 86
D-fructose
0.00055 - 212
D-mannitol
64
D-xylulose
Aspergillus fumigatus
-
pH 7.1, 25C
60
L-sorbitol
Aspergillus fumigatus
-
pH 10.0, 25C
3 - 24
NAD(P)H
0.04 - 40
NAD+
0.00045 - 61
NADH
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.99
D-arabinitol
Aspergillus fumigatus
-
pH 10.0, 25C
2268
0.000026 - 250
D-fructose
0.000015 - 100
D-mannitol
39
D-xylulose
Aspergillus fumigatus
-
pH 7.1, 25C
715
0.088
L-sorbitol
Aspergillus fumigatus
-
pH 10.0, 25C
5292
1.1
L-sorbose
Aspergillus fumigatus
-
pH 7.1, 25C
685
0.0024 - 400
NAD+
0.019 - 910
NADH
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.6 - 5.7
ADP
2.9 - 4.8
AMP
1356
D-fructose
-
-
24.6
D-mannitol
0.003 - 0.6
NAD+
0.061 - 0.08
NADH
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.003
-
cells grown on D-glucose, crude extracts
0.012
-
cells grown on D-mannitol + glucose, crude extracts
0.015
-
cells grown on D-ribose, crude extracts
0.05
-
41C, pH 9.0
0.243
-
cells grown on D-mannitol, crude extracts
14
-
crude extract, at 25C, using 100 mM Tris-HCl buffer, pH 7.1
33
-
after 6.7fold purification, at 25C, using 100 mM Tris-HCl buffer, pH 7.1
46
-
histidine-tagged recombinant protein
54
purified enzyme at 80C with D-fructose as the substrate and NADH as the cofactor; purified enzyme, at 80C with fructose as the substrate and NADH as the cofactor
63
-
MDH expressed in Escherichia coli strain BL21 (DE3) plysS, with fructose as substrate
68.3
-
recombinant protein
additional information
-
the activity for inositol, sorbitol, galactitol, ribitol, xylitol, arabitol and erythritol is very low
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.4
-
reduction of D-fructose
5.5
-
for reduction of D-fructose; optimal for D-fructose reduction
5.5 - 6
5.8
-
reduction of D-fructose
6 - 7.2
6 - 7.2
7
-
D-fructose reduction, recombinant protein
8.3 - 8.6
optimally oxidizes L-mannitol between pH 8.3 and 8.6
8.9 - 10
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 7
-
pH 4.5: about 80% of maximal activity, pH 7.0: about 60% of maximal activity, reduction of D-fructose
6 - 6.5
-
not active below, D-mannitol oxidation
6.8 - 8.3
-
pH 6.8: about 20% of maximal activity, pH 8.3: about 55% of maximal activity, oxidation of D-mannitol
8 - 9.5
-
-
8
-
not active above, D-fructose reduction
9 - 9.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at, both reaction directions
35
-
reduction of D-fructose
40
-
for oxidation of D-mannitol; optimal for D-mannitol oxidation
50
-
for reduction of D-fructose; optimal for D-fructose reduction
90 - 100
most active around 95C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 45
-
-
20 - 40
-
20C: about 85% of maximal activity, 40C: about 70% of maximal activity, reduction of D-fructose
90 - 120
MtDH retains 63% of its activity at 120C but shows no detectable activity at 25C
95 - 120
retains 63% of its activity at 120C but shows no detectable activity at room temperature
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
36000
-
estimated molecular mass from sequence of cDNA
45000
-
recombinant protein, gel filtration
47200
-
sucrose density gradient centrifugation
54000
-
recombinant protein, SDS-PAGE
54490
-
calculated from gene sequence
58000
-
recombinant enzyme, SDS-PAGE
64500
-
sucrose density gradient centrifugation
120000 - 134000
gel filtration
120000 - 143000
gel filtration or analytical ultracentrifugation
120000
ultracentrifugation
132000 - 138000
-
gel filtration, sucrose density gradient centrifugation
133000
-
gel filtration
155000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 55000, SDS-PAGE
monomer
octamer
gel filtration at room temperature, MtDH in solution
tetramer
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
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 6.5
-
-
287257, 287258
6 - 6.5
-
highest stability
287241
8.5
TM0298 is sensitive to proteolytic degradation in purification protocols at pH 8.5
695814
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
-
not stable above
25
-
half-life 3.6 h
30
-
half-life 0.42 h
30 - 40
-
The enzyme is very stable at 30C. The half life time at 40C is 35 min for D-mannitol oxidation.
50
-
heat inactivation, 50% loss of activity after 30 min incubation
80
half-life: 57 min; half-lives of 57 min
95
half-life: 6 min; half-lives of 6 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
stabilized with 0.5 mM EDTA, 1% bovine serum albumin
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-10C, 1 month, 50% loss of activity
-
-16C, 0.02 M sodium acetate buffer pH 6.0, 1 mM 2-mercaptoethanol, several weeks
-
-20C, 10 mM dithiothreitol, 45 days
-
3C, crystalline enzyme in ammonium sulfate, 2 months
-
4C or 20C, 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
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by heat treatment at 85C and on Ni-NTA column; Ni-NTA agarose column chromatography
by Ni2+-NTA affinity chromatography
-
Co2+ chelating Sepharose column chromatography and Superdex 200 gel filtration
-
HisBind affinity chromatography
-
histidine-tagged recombinant protein, expressed in Escherichia coli
-
mutants purified to apparent homogeneity
-
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
-
recombinant protein
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells; 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)
expressed in Escherichia coli JM109 cells
-
expressed in Escherichia coli strain BL21pLysS
-
expressed in Escherichia coli; expression of wild-type and mutant enzymes in Escherichia coli strain JM109
-
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 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)
-
MtDH is expressed in Escherichia coli BL21(DE3)
subcloned into vector pDEST110 and overexpressed in different strains of Escherichia coli (BL21 (DE3) plysS, JM109, Origami(DE3) or M15)
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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); utilizes NAD(H) and NADP(H) with similar catalytic efficiencies. Uses NADP(H) almost as well as wild-type enzyme uses NAD(H)
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
-
mutant enzyme displays catalytic efficiency for NAD+-dependent oxidation of D-mannitol 300fold below the wild-type value
K295M
-
2000000fold lower turnover number for D-mannitol oxidation at pH 10.0 than the wild-type enzyme
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 enzymeNAD+-mannitol complex
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 enzymeNAD+-mannitol complex
N300A
-
mutant enzyme displays catalytic efficiency for NAD+-dependent oxidation of D-mannitol 1000fold below the wild-type value
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
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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
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
the recombinant enzyme expressed in Bacillus megaterium is useful in production of D-mannitol using a resting cell biotransformation approach
Show AA Sequence (294 entries)
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