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Literature summary for 1.1.1.67 extracted from

  • Song, S.H.; Ahluwalia, N.; Leduc, Y.; Delbaere, L.T.; Vieille, C.
    Thermotoga maritima TM0298 is a highly thermostable mannitol dehydrogenase (2008), Appl. Microbiol. Biotechnol., 81, 485-495.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Thermotoga maritima
MtDH is expressed in Escherichia coli BL21(DE3) Limosilactobacillus reuteri
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) Thermotoga maritima

Inhibitors

Inhibitors Comment Organism Structure
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 Thermotoga maritima
mannitol
-
Thermotoga maritima

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.037
-
NADH at 60°C and pH 6.1 Thermotoga maritima
0.037
-
NADH at 60°C, pH 6.1 Thermotoga maritima
0.048
-
NADH at 80°C and pH 6.1 Thermotoga maritima
0.048
-
NADH at 80°C, pH 6.1 Thermotoga maritima
0.14
-
NAD+ at 80°C and pH 8.3 Thermotoga maritima
0.14
-
NAD+ at 80°C, pH 8.3 Thermotoga maritima
0.17
-
NADPH at 80°C and pH 6.1 Thermotoga maritima
0.17
-
NADPH at 80°C, pH 6.1 Thermotoga maritima
5.51
-
D-mannitol at 80°C and pH 8.3 Thermotoga maritima
5.51
-
D-mannitol at 80°C, pH 8.3 Thermotoga maritima
7.5
-
NADP+ at 80°C and pH 8.3 Thermotoga maritima
7.5
-
NADP+ at 80°C, pH 8.3 Thermotoga maritima
13.23
-
D-mannitol at 60°C and pH 6.1 Thermotoga maritima
13.23
-
D-mannitol at 60°C, pH 6.1 Thermotoga maritima
50
-
D-fructose at 60°C and pH 6.1 Thermotoga maritima
50
-
D-fructose at 60°C, pH 6.1 Thermotoga maritima
50.97
-
D-fructose at 80°C and pH 6.1 Thermotoga maritima
50.97
-
D-fructose at 80°C, pH 6.1 Thermotoga maritima

Metals/Ions

Metals/Ions Comment Organism Structure
Co2+
-
Thermotoga maritima
Mn2+
-
Thermotoga maritima
additional information 20 mM MgCl2 or CaCl2 do not increase the activity of the EDTA-treated enzyme Thermotoga maritima
Zn2+ contains 0.69 mol of Zn2+ per subunit of enzyme Thermotoga maritima
Zn2+ MtDH contains a single catalytic zinc per subunit Thermotoga maritima

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
34000
-
4 * 34000, SDS-PAGE Thermotoga maritima
120000
-
ultracentrifugation Thermotoga maritima
120000 143000 gel filtration or analytical ultracentrifugation Thermotoga maritima
120000 134000 gel filtration Thermotoga maritima

Organism

Organism UniProt Comment Textmining
Limosilactobacillus reuteri Q6ECH5
-
-
Thermotoga maritima Q9WYD4
-
-
Thermotoga maritima MSB8 / DSM 3109 / ATCC 43589 Q9WYD4
-
-

Purification (Commentary)

Purification (Comment) Organism
by heat treatment at 85°C and on Ni-NTA column Thermotoga maritima
Ni-NTA agarose column chromatography Thermotoga maritima

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
54
-
purified enzyme at 80°C with D-fructose as the substrate and NADH as the cofactor Thermotoga maritima
54
-
purified enzyme, at 80°C with fructose as the substrate and NADH as the cofactor Thermotoga maritima

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
D-fructose + NADPH + H+ also active on fructose with NADPH Thermotoga maritima D-mannitol + NADP+
-
?
D-fructose + NADPH + H+ also active on fructose with NADPH Thermotoga maritima MSB8 / DSM 3109 / ATCC 43589 D-mannitol + NADP+
-
?
D-mannitol + NAD(P)+
-
Thermotoga maritima D-fructose + NAD(P)H + H+
-
r
D-mannitol + NAD(P)+
-
Thermotoga maritima MSB8 / DSM 3109 / ATCC 43589 D-fructose + NAD(P)H + H+
-
r
D-mannitol + NAD+ 100% activity Thermotoga maritima D-fructose + NADH + H+
-
r
D-mannitol + NAD+ 100% activity Thermotoga maritima MSB8 / DSM 3109 / ATCC 43589 D-fructose + NADH + H+
-
r
D-tagatose + NAD(P)+ 29% relative activity compared to D-fructose Thermotoga maritima ? + NAD(P)H
-
?
D-tagatose + NAD+ 29% relative activity on D-tagatose compared to 100% activity on D-fructose Thermotoga maritima ?
-
?
D-xylulose + NAD(P)+ 18% relative activity compared to D-fructose Thermotoga maritima ? + NAD(P)H
-
?
D-xylulose + NAD+ 18% relative activity on D-xylulose compared to 100% activity on D-fructose Thermotoga maritima ?
-
?
L-sorbose + NAD(P)+ 5% relative activity compared to D-fructose Thermotoga maritima ? + NAD(P)H
-
?
L-sorbose + NADH + H+ 5% relative activity on L-sorbose compared to 100% activity on D-fructose Thermotoga maritima L-sorbitol + NAD+
-
?
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 Thermotoga maritima ?
-
?
additional information TM0298 shows no detectable activity on glucose, xylose, threonine, arabinose, acetaldehyde, 2-butanone, sorbitol, xylitol, ethanol, or 2-butanol Thermotoga maritima ?
-
?
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 Thermotoga maritima MSB8 / DSM 3109 / ATCC 43589 ?
-
?
additional information TM0298 shows no detectable activity on glucose, xylose, threonine, arabinose, acetaldehyde, 2-butanone, sorbitol, xylitol, ethanol, or 2-butanol Thermotoga maritima MSB8 / DSM 3109 / ATCC 43589 ?
-
?

Subunits

Subunits Comment Organism
dimer gel filtration at room temperature, MtDH in solution Limosilactobacillus reuteri
octamer gel filtration at room temperature, MtDH in solution Limosilactobacillus reuteri
tetramer 4 * 34000, SDS-PAGE Thermotoga maritima
tetramer gel filtration at room temperature, MtDH in solution Limosilactobacillus reuteri
tetramer gel filtration or analytical ultracentrifugation, MtDH in solution Thermotoga maritima

Synonyms

Synonyms Comment Organism
MtDH
-
Thermotoga maritima
MtDH
-
Limosilactobacillus reuteri
TM0298
-
Thermotoga maritima

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
90 100 most active around 95°C Thermotoga maritima
95
-
-
Thermotoga maritima

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
90 120 MtDH retains 63% of its activity at 120°C but shows no detectable activity at 25°C Thermotoga maritima
95 120 retains 63% of its activity at 120°C but shows no detectable activity at room temperature Thermotoga maritima

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
80
-
half-life: 57 min Thermotoga maritima
80
-
half-lives of 57 min Thermotoga maritima
95
-
half-life: 6 min Thermotoga maritima
95
-
half-lives of 6 min Thermotoga maritima

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
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 Thermotoga maritima
5.5 6 optimally reduces L-fructose at pH values between pH 5.5 and 6.0 Thermotoga maritima
8.3 8.6 optimally oxidizes L-mannitol between pH 8.3 and 8.6 Thermotoga maritima

pH Stability

pH Stability pH Stability Maximum Comment Organism
8.5
-
TM0298 is sensitive to proteolytic degradation in purification protocols at pH 8.5 Thermotoga maritima

Cofactor

Cofactor Comment Organism Structure
NAD(P)+ its catalytic efficiency is 33times higher with NAD+ than with NADP+ Thermotoga maritima
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 Thermotoga maritima
NAD+ although MtDH has a higher Vmax with NADPH than with NADH, its catalytic efficiency is 33 times higher with NAD+ than with NADP+ Thermotoga maritima
NADH although MtDH has a higher Vmax with NADPH than with NADH, its catalytic efficiency is 2.2times higher with NADH than with NADPH Thermotoga maritima

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
99
-
mannitol
-
Thermotoga maritima