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Literature summary for 1.1.1.2 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
Search for more literature for 1.1.1.2

Activating Compound

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

Cloned(Commentary)

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

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 [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

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

Organic Solvent Stability

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

Organism UniProt Comment Textmining
Thermococcus guaymasensis F8S123
-
-

Oxidation Stability

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 (Commentary)

Purification (Comment) Organism
-
 Thermococcus guaymasensis

Substrates and Products (Substrate)

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

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

Temperature Optimum [°C]

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 [°C]

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 [1/s]

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

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

Cofactor

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

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 [mM/s]

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