Activating Compound | Comment | Organism | Structure |
---|---|---|---|
Isopropanol | highly efficient reduction of ketones by coupling with excess isopropanol to regenerate NADPH | Thermococcus guaymasensis |
Cloned (Comment) | Organism |
---|---|
the gene encoding the enzyme is cloned and sequenced, it shows high sequence homology to zinc-containing alcohol dehydrogenases and L-threonine dehydrogenases with binding motifs of catalytic zinc and NADP+ | Thermococcus guaymasensis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
Cd2+ | 1 mM, complete inhibition | Thermococcus guaymasensis | |
Cu2+ | 1 mM, complete inhibition | Thermococcus guaymasensis | |
EDTA | 1 mM, slightly decreased the enzyme activity | Thermococcus guaymasensis | |
Fe2+ | 1 mM, 10-20% decrease in activity | Thermococcus guaymasensis | |
Hg2+ | 1 mM, complete inhibition | Thermococcus guaymasensis | |
Ni2+ | 1 mM, 10-20% decrease in activity | Thermococcus guaymasensis | |
Zn2+ | 1 mM, complete inhibition | Thermococcus guaymasensis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.011 | - |
NADPH | cosubstrate: 2-butanone, pH 7.5, 80°C | Thermococcus guaymasensis | |
0.21 | - |
diacetyl | cosubstrate: NADPH, pH 7.5, 80°C | Thermococcus guaymasensis | |
0.31 | - |
2-butanone | cosubstrate: NADPH, pH 7.5, 80°C | Thermococcus guaymasensis | |
0.32 | - |
acetoin | cosubstrate: NADPH, pH 7.5, 80°C | Thermococcus guaymasensis | |
0.38 | - |
2-butanol | cosubstrate: NADP+, pH 7.5, 80°C | Thermococcus guaymasensis | |
0.4 | - |
NADP+ | cosubstrate: 2-butanol, pH 7.5, 80°C | Thermococcus guaymasensis | |
15.2 | - |
(2R,3R)-2,3-butanediol | cosubstrate: NADP+, pH 7.5, 80°C | Thermococcus guaymasensis | |
19.3 | - |
meso-2,3-butanediol | cosubstrate: NADP+, pH 7.5, 80°C | Thermococcus guaymasensis | |
246 | - |
(2S,3S)-2,3-butanediol | cosubstrate: NADP+, pH 7.5, 80°C | Thermococcus guaymasensis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Ca2+ | 1 mM, less than 15% increase of activity | Thermococcus guaymasensis | |
Co2+ | 1 mM, less than 15% increase of activity | Thermococcus guaymasensis | |
Mg2+ | 1 mM, less than 15% increase of activity | Thermococcus guaymasensis | |
Mn2+ | 1 mM, less than 15% increase of activity | Thermococcus guaymasensis | |
Zinc | contains 0.9 gatoms of zinc per subunit | Thermococcus guaymasensis |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
40000 | - |
4 * 40000, SDS-PAGE | Thermococcus guaymasensis |
135000 | - |
gel filtration | Thermococcus guaymasensis |
Organic Solvent | Comment | Organism |
---|---|---|
isopropanol | solvent-tolerant enzyme, highly efficient reduction of ketones by coupling with excess isopropanol to regenerate NADPH | Thermococcus guaymasensis |
Methanol | the purified enzyme does not oxidize methanol. The enzyme retains almost its full activity when the concentration of methanol is up to 5% (v/v). When the concentration of methanol is 40% (v/v) in the assay mixture, the enzyme activities remain at 30% and 15% of the full activity on both oxidation and reduction, respectively | Thermococcus guaymasensis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermococcus guaymasensis | F8S123 | - |
- |
Oxidation Stability | Organism |
---|---|
the enzyme is oxygen sensitive, although it is much more resistant to oxidation than iron-containing alcohol dehydrogenases. The time required to decrease 50% of the full activity upon exposure to the air (t1/2) is about 4 h, and such inactivation is slightly decreased in the presence of 2 mM dithiothreitol | Thermococcus guaymasensis |
Purification (Comment) | Organism |
---|---|
- |
Thermococcus guaymasensis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(2R,3R)-2,3-butanediol + NADP+ | the enzyme shows higher oxidation activities on the (2R,3R)-(-)-2,3-butanediol and meso-2,3-butanediol than on the (2S,3S)-(+)-2,3-butanediol, indicating that the enzyme predominantly oxidizes the R-hydroxyl group of 2,3-butanediol and minorly functions on the S-hydroxyl group. When meso-2,3-butanediol is oxidized, (S)-2-acetoin is the predominant product, with an enantiomeric excess (ee) of 88%, while oxidation of (2R,3R)-2,3-butanediol results in the production of (R)-2-acetoin with higher stereoselectivity (94% ee) | Thermococcus guaymasensis | (R)-2-acetoin + NADPH + H+ | - |
r | |
(2S,3S)-2,3-butanediol + NADP+ | the enzyme shows higher oxidation activities on the (2R,3R)-(-)-2,3-butanediol and meso-2,3-butanediol than on the (2S,3S)-(+)-2,3-butanediol, indicating that the enzyme predominantly oxidizes the R-hydroxyl group of 2,3-butanediol and minorly functions on the S-hydroxyl group. When meso-2,3-butanediol is oxidized, (S)-2-acetoin is the predominant product, with an enantiomeric excess (ee) of 88%, while oxidation of meso-2,3-butanediol results in the production of (R)-2-acetoin with higher stereoselectivity (94% ee) | Thermococcus guaymasensis | (S)-2-acetoin + NADPH + H+ | - |
r | |
1,2-butanediol + NADP+ | 25.6% of the activity compared to the substrate 2-butanol | Thermococcus guaymasensis | ? + NADPH + H+ | - |
? | |
1,3-butanediol + NADP+ | 35.4% of the activity compared to the substrate 2-butanol | Thermococcus guaymasensis | ? + NADPH + H+ | - |
? | |
1-butanol + NADP+ | 5.1% of the activity compared to the substrate 2-butanol | Thermococcus guaymasensis | butyraldehyde + NADPH + H+ | - |
r | |
1-propanol + NADP+ | 15.1% of the activity compared to the substrate 2-butanol | Thermococcus guaymasensis | propanal + NADPH + H+ | - |
? | |
2,4-pentanediol + NADP+ | 9.2% of the activity compared to the substrate 2-butanol | Thermococcus guaymasensis | ? + NADPH + H+ | - |
? | |
2-butanol + NADP+ | the enzyme does not possess stereoselectivity on 2-butanol or 2-butanone as substrate | Thermococcus guaymasensis | 2-butanone + NADPH + H+ | - |
r | |
2-butanone + NADPH + H+ | the enzyme does not possess stereoselectivity on 2-butanol or 2-butanone as substrate | Thermococcus guaymasensis | 2-butanol + NADP+ | - |
r | |
2-pentanol + NADP+ | 66.3% of the activity compared to the substrate 2-butanol | Thermococcus guaymasensis | 2-pentanone + NADPH + H+ | - |
r | |
2-pentanone + NADPH + H+ | 86.2% of the activity compared to the substrate 2-butanone | Thermococcus guaymasensis | 2-pentanol + NADP+ | - |
r | |
2-propanol + NADP+ | 88% of the activity compared to the substrate 2-butanol | Thermococcus guaymasensis | acetone + NADPH + H+ | - |
r | |
acetaldehyde + NADPH + H+ | - |
Thermococcus guaymasensis | ethanol + NADP+ | - |
r | |
acetoin + NADPH + H+ | 93% of the activity compared to the substrate 2-butanone. The racemic R/S-acetoin is used since neither R- nor S-acetoin is commercially available. Consistently, the reduction of racemic R/S-acetoin formed (2R,3R)-2,3-butanediol (presumably from R-acetoin) and meso-2,3-butanediol (presumably from S-acetoin) with extremely high stereoselectivity (99% ee over (2S,3S)-(+)-2,3-butanediol) | Thermococcus guaymasensis | 2,3-butanediol + NADP+ | - |
r | |
acetone + NADPH + H+ | 149% of the activity compared to the substrate 2-butanone | Thermococcus guaymasensis | 2-propanol + NADP+ | - |
r | |
butyraldehyde + NADPH + H+ | 112.4% of the activity compared to the substrate 2-butanone | Thermococcus guaymasensis | 1-butanol + NADP+ | - |
r | |
diacetyl + NADPH + H+ | the enzyme can not oxidize acetoin to diacetyl | Thermococcus guaymasensis | acetoin + NADP+ | - |
ir | |
ethanol + NADP+ | 5.7% of the activity compared to the substrate 2-butanol | Thermococcus guaymasensis | acetaldehyde + NADPH + H+ | - |
r | |
meso-2,3-butanediol + NADP+ | the enzyme shows higher oxidation activities on the (2R,3R)-(-)-2,3-butanediol and meso-2,3-butanediol than on the (2S,3S)-(+)-2,3-butanediol, indicating that the enzyme predominantly oxidizes the R-hydroxyl group of 2,3-butanediol and minorly functions on the S-hydroxyl group. When meso-2,3-butanediol is oxidized, (S)-2-acetoin is the predominant product, with an enantiomeric excess (ee) of 88%, while oxidation of meso-2,3-butanediol results in the production of (R)-2-acetoin with higher stereoselectivity (94% ee) | Thermococcus guaymasensis | (S)-2-acetoin + NADPH + H+ | - |
r | |
additional information | the enzyme shows higher activities with the use of secondary alcohols such as 2-butanol and 2-pentanol as substrates, suggesting that the enzyme is a primary-secondary alcohol dehydrogenase. Alcohols oxidized at an acticity of less than 5% compared to the substrate 2-butanol: 1-pentanol, glycerol, 1,2-pentanediol. No oxidation of methanol, 1,4-butanediol, 1,5-pentanediol, ethanediol, phenylethanol, L-serine, L-threonine, acetoin | Thermococcus guaymasensis | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
homotetramer | 4 * 40000, SDS-PAGE | Thermococcus guaymasensis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
95 | - |
activity increases along with the elevated temperatures up to 95°C | Thermococcus guaymasensis |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
80 | - |
t1/2: 70 h | Thermococcus guaymasensis |
95 | - |
t1/2: 24 h | Thermococcus guaymasensis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
142 | - |
acetoin | cosubstrate: NADPH, pH 7.5, 80°C | Thermococcus guaymasensis | |
158 | - |
NADPH | cosubstrate: 2-butanone, pH 7.5, 80°C | Thermococcus guaymasensis | |
190 | - |
2-butanone | cosubstrate: NADPH, pH 7.5, 80°C | Thermococcus guaymasensis | |
202 | - |
diacetyl | cosubstrate: NADPH, pH 7.5, 80°C | Thermococcus guaymasensis | |
512 | - |
(2S,3S)-2,3-butanediol | cosubstrate: NADP+, pH 7.5, 80°C | Thermococcus guaymasensis | |
740 | - |
(2R,3R)-2,3-butanediol | cosubstrate: NADP+, pH 7.5, 80°C | Thermococcus guaymasensis | |
833 | - |
2-butanol | cosubstrate: NADP+, pH 7.5, 80°C | Thermococcus guaymasensis | |
952 | - |
meso-2,3-butanediol | cosubstrate: NADP+, pH 7.5, 80°C | Thermococcus guaymasensis | |
1333 | - |
NADP+ | cosubstrate: 2-butanol, pH 7.5, 80°C | Thermococcus guaymasensis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
2-butanone reduction | Thermococcus guaymasensis |
10.5 | - |
2-butanol oxidation | Thermococcus guaymasensis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADP+ | Km values and catalytic efficiency for NADPH are 40times lower and 5times higher than those for NADP+, respectively | Thermococcus guaymasensis | |
NADPH | Km values and catalytic efficiency for NADPH are 40times lower and 5times higher than those for NADP+, respectively | Thermococcus guaymasensis |
General Information | Comment | Organism |
---|---|---|
physiological function | the physiological roles of the enzyme are proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation | Thermococcus guaymasensis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.08 | - |
(2S,3S)-2,3-butanediol | cosubstrate: NADP+, pH 7.5, 80°C | Thermococcus guaymasensis | |
49 | - |
meso-2,3-butanediol | cosubstrate: NADP+, pH 7.5, 80°C | Thermococcus guaymasensis | |
49 | - |
(2R,3R)-2,3-butanediol | cosubstrate: NADP+, pH 7.5, 80°C | Thermococcus guaymasensis | |
444 | - |
acetoin | cosubstrate: NADPH, pH 7.5, 80°C | Thermococcus guaymasensis | |
613 | - |
2-butanone | cosubstrate: NADPH, pH 7.5, 80°C | Thermococcus guaymasensis | |
962 | - |
diacetyl | cosubstrate: NADPH, pH 7.5, 80°C | Thermococcus guaymasensis | |
2192 | - |
2-butanol | cosubstrate: NADP+, pH 7.5, 80°C | Thermococcus guaymasensis | |
3333 | - |
NADP+ | cosubstrate: 2-butanol, pH 7.5, 80°C | Thermococcus guaymasensis | |
14360 | - |
NADPH | cosubstrate: 2-butanone, pH 7.5, 80°C | Thermococcus guaymasensis |