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Literature summary extracted from

  • Ying, X.; Ma, K.
    Characterization of a zinc-containing alcohol dehydrogenase with stereoselectivity from the hyperthermophilic archaeon Thermococcus guaymasensis (2011), J. Bacteriol., 183, 3009-3019.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.1.1.2 Isopropanol highly efficient reduction of ketones by coupling with excess isopropanol to regenerate NADPH Thermococcus guaymasensis

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.2 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

EC Number Inhibitors Comment Organism Structure
1.1.1.2 Cd2+ 1 mM, complete inhibition Thermococcus guaymasensis
1.1.1.2 Cu2+ 1 mM, complete inhibition Thermococcus guaymasensis
1.1.1.2 EDTA 1 mM, slightly decreased the enzyme activity Thermococcus guaymasensis
1.1.1.2 Fe2+ 1 mM, 10-20% decrease in activity Thermococcus guaymasensis
1.1.1.2 Hg2+ 1 mM, complete inhibition Thermococcus guaymasensis
1.1.1.2 Ni2+ 1 mM, 10-20% decrease in activity Thermococcus guaymasensis
1.1.1.2 Zn2+ 1 mM, complete inhibition Thermococcus guaymasensis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.2 0.011
-
NADPH cosubstrate: 2-butanone, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 0.21
-
diacetyl cosubstrate: NADPH, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 0.31
-
2-butanone cosubstrate: NADPH, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 0.32
-
acetoin cosubstrate: NADPH, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 0.38
-
2-butanol cosubstrate: NADP+, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 0.4
-
NADP+ cosubstrate: 2-butanol, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 15.2
-
(2R,3R)-2,3-butanediol cosubstrate: NADP+, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 19.3
-
meso-2,3-butanediol cosubstrate: NADP+, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 246
-
(2S,3S)-2,3-butanediol cosubstrate: NADP+, pH 7.5, 80°C Thermococcus guaymasensis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.1.1.2 Ca2+ 1 mM, less than 15% increase of activity Thermococcus guaymasensis
1.1.1.2 Co2+ 1 mM, less than 15% increase of activity Thermococcus guaymasensis
1.1.1.2 Mg2+ 1 mM, less than 15% increase of activity Thermococcus guaymasensis
1.1.1.2 Mn2+ 1 mM, less than 15% increase of activity Thermococcus guaymasensis
1.1.1.2 Zinc contains 0.9 gatoms of zinc per subunit Thermococcus guaymasensis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.1.1.2 40000
-
4 * 40000, SDS-PAGE Thermococcus guaymasensis
1.1.1.2 135000
-
gel filtration Thermococcus guaymasensis

Organic Solvent Stability

EC Number Organic Solvent Comment Organism
1.1.1.2 isopropanol solvent-tolerant enzyme, highly efficient reduction of ketones by coupling with excess isopropanol to regenerate NADPH Thermococcus guaymasensis
1.1.1.2 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

EC Number Organism UniProt Comment Textmining
1.1.1.2 Thermococcus guaymasensis F8S123
-
-

Oxidation Stability

EC Number Oxidation Stability Organism
1.1.1.2 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 (Commentary)

EC Number Purification (Comment) Organism
1.1.1.2
-
Thermococcus guaymasensis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.2 (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
1.1.1.2 (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
1.1.1.2 1,2-butanediol + NADP+ 25.6% of the activity compared to the substrate 2-butanol Thermococcus guaymasensis ? + NADPH + H+
-
?
1.1.1.2 1,3-butanediol + NADP+ 35.4% of the activity compared to the substrate 2-butanol Thermococcus guaymasensis ? + NADPH + H+
-
?
1.1.1.2 1-butanol + NADP+ 5.1% of the activity compared to the substrate 2-butanol Thermococcus guaymasensis butyraldehyde + NADPH + H+
-
r
1.1.1.2 1-propanol + NADP+ 15.1% of the activity compared to the substrate 2-butanol Thermococcus guaymasensis propanal + NADPH + H+
-
?
1.1.1.2 2,4-pentanediol + NADP+ 9.2% of the activity compared to the substrate 2-butanol Thermococcus guaymasensis ? + NADPH + H+
-
?
1.1.1.2 2-butanol + NADP+ the enzyme does not possess stereoselectivity on 2-butanol or 2-butanone as substrate Thermococcus guaymasensis 2-butanone + NADPH + H+
-
r
1.1.1.2 2-butanone + NADPH + H+ the enzyme does not possess stereoselectivity on 2-butanol or 2-butanone as substrate Thermococcus guaymasensis 2-butanol + NADP+
-
r
1.1.1.2 2-pentanol + NADP+ 66.3% of the activity compared to the substrate 2-butanol Thermococcus guaymasensis 2-pentanone + NADPH + H+
-
r
1.1.1.2 2-pentanone + NADPH + H+ 86.2% of the activity compared to the substrate 2-butanone Thermococcus guaymasensis 2-pentanol + NADP+
-
r
1.1.1.2 2-propanol + NADP+ 88% of the activity compared to the substrate 2-butanol Thermococcus guaymasensis acetone + NADPH + H+
-
r
1.1.1.2 acetaldehyde + NADPH + H+
-
Thermococcus guaymasensis ethanol + NADP+
-
r
1.1.1.2 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
1.1.1.2 acetone + NADPH + H+ 149% of the activity compared to the substrate 2-butanone Thermococcus guaymasensis 2-propanol + NADP+
-
r
1.1.1.2 butyraldehyde + NADPH + H+ 112.4% of the activity compared to the substrate 2-butanone Thermococcus guaymasensis 1-butanol + NADP+
-
r
1.1.1.2 diacetyl + NADPH + H+ the enzyme can not oxidize acetoin to diacetyl Thermococcus guaymasensis acetoin + NADP+
-
ir
1.1.1.2 ethanol + NADP+ 5.7% of the activity compared to the substrate 2-butanol Thermococcus guaymasensis acetaldehyde + NADPH + H+
-
r
1.1.1.2 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
1.1.1.2 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

EC Number Subunits Comment Organism
1.1.1.2 homotetramer 4 * 40000, SDS-PAGE Thermococcus guaymasensis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.2 95
-
activity increases along with the elevated temperatures up to 95°C Thermococcus guaymasensis

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.1.1.2 80
-
t1/2: 70 h Thermococcus guaymasensis
1.1.1.2 95
-
t1/2: 24 h Thermococcus guaymasensis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.2 142
-
acetoin cosubstrate: NADPH, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 158
-
NADPH cosubstrate: 2-butanone, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 190
-
2-butanone cosubstrate: NADPH, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 202
-
diacetyl cosubstrate: NADPH, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 512
-
(2S,3S)-2,3-butanediol cosubstrate: NADP+, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 740
-
(2R,3R)-2,3-butanediol cosubstrate: NADP+, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 833
-
2-butanol cosubstrate: NADP+, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 952
-
meso-2,3-butanediol cosubstrate: NADP+, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 1333
-
NADP+ cosubstrate: 2-butanol, pH 7.5, 80°C Thermococcus guaymasensis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.2 7.5
-
2-butanone reduction Thermococcus guaymasensis
1.1.1.2 10.5
-
2-butanol oxidation Thermococcus guaymasensis

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.2 NADP+ Km values and catalytic efficiency for NADPH are 40times lower and 5times higher than those for NADP+, respectively Thermococcus guaymasensis
1.1.1.2 NADPH Km values and catalytic efficiency for NADPH are 40times lower and 5times higher than those for NADP+, respectively Thermococcus guaymasensis

General Information

EC Number General Information Comment Organism
1.1.1.2 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 [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.2 2.08
-
(2S,3S)-2,3-butanediol cosubstrate: NADP+, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 49
-
meso-2,3-butanediol cosubstrate: NADP+, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 49
-
(2R,3R)-2,3-butanediol cosubstrate: NADP+, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 444
-
acetoin cosubstrate: NADPH, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 613
-
2-butanone cosubstrate: NADPH, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 962
-
diacetyl cosubstrate: NADPH, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 2192
-
2-butanol cosubstrate: NADP+, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 3333
-
NADP+ cosubstrate: 2-butanol, pH 7.5, 80°C Thermococcus guaymasensis
1.1.1.2 14360
-
NADPH cosubstrate: 2-butanone, pH 7.5, 80°C Thermococcus guaymasensis