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Literature summary for 1.1.1.B4 extracted from

  • Pennacchio, A.; Pucci, B.; Secundo, F.; La Cara, F.; Rossi, M.; Raia, C.A.
    Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus (2008), Appl. Environ. Microbiol., 74, 3949-3958.
    View publication on PubMedView publication on EuropePMC

Activating Compound

Activating Compound Comment Organism Structure
o-phenanthroline activates to 108% activity compared to the control activity at 0.1 mM and to 109% at 1 mM Thermus thermophilus

Application

Application Comment Organism
synthesis the enzym is useful in production of chiral compounds for organic synthesis Thermus thermophilus

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli strain BL21(DE3) Thermus thermophilus

Inhibitors

Inhibitors Comment Organism Structure
1-butyl-3-methylimidazolium tetrafluoroborate 65% inhibition at 2 mM Thermus thermophilus
Ca2+ 10% inhibition at 1 mM and 30% at 100 mM Thermus thermophilus
Cu2+ 23% inhibition at 1 mM Thermus thermophilus
Hg2+ 67% inhibition at 1 mM Thermus thermophilus
iodoacetate 5% inhibition at 1 mM Thermus thermophilus
Mg2+ 33% inhibition at 100 mM Thermus thermophilus
additional information no effects by 5 mM 2-mercaptoethanol Thermus thermophilus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information kinetics and substrate specificity, overview Thermus thermophilus
0.035
-
NADH pH 6.0, 65°C, recombinant enzyme, reduction reaction Thermus thermophilus
0.24
-
NAD+ pH 6.0, 65°C, recombinant enzyme, reduction reaction Thermus thermophilus
1
-
ethylbenzoylformate pH 6.0, 65°C, recombinant enzyme, reduction reaction Thermus thermophilus
11.2
-
2,2,2-trifluoroacetophenone pH 6.0, 65°C, recombinant enzyme, reduction reaction Thermus thermophilus

Metals/Ions

Metals/Ions Comment Organism Structure
EDTA activates to 127% activity compared to the control activity at 1 mM and to 163% at 10 mM Thermus thermophilus
K+ activates to 167% activity compared to the control activity at 1 mM and to 193% at 100 mM Thermus thermophilus
Li+ activates to 127% activity compared to the control activity at 1 mM and to 178% at 100 mM Thermus thermophilus
Na+ activates to 179 at 100 mM Thermus thermophilus
Zn2+ activates to 114% activity compared to the control activity at 1 mM Thermus thermophilus

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
26000
-
4 * 26961, recombinant enzyme, mass spectrometry, 4 * 26000, recombinant enzyme, SDS-PAGE Thermus thermophilus
26961
-
4 * 26961, recombinant enzyme, mass spectrometry, 4 * 26000, recombinant enzyme, SDS-PAGE Thermus thermophilus
71000
-
about, recombinant enzyme, gel filtration in absence of NaCl Thermus thermophilus
105000
-
about, recombinant enzyme, gel filtration in presence of 0.15 M NaCl Thermus thermophilus

Organic Solvent Stability

Organic Solvent Comment Organism
additional information the enzyme shows a good tolerance to common organic solvents Thermus thermophilus

Organism

Organism UniProt Comment Textmining
Thermus thermophilus
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant enzyme 11fold to homogeneity from Escherichia coli strain BL21(DE3) by heat denaturation of host proteins, anion exchange chromatography and gel filtration Thermus thermophilus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(R)-(trifluoromethyl)benzyl alcohol + NADH + H+ 93% ethanoselective reaction Thermus thermophilus ?
-
r
2,2,2-trifluoroacetophenone + NADH + H+ completely enantioselective reaction Thermus thermophilus (1R)-2,2,2-trifluoro-1-phenylethanol + NAD+
-
r
ethyl (R)-mandelate + NADH + H+ 95% ethanoselective reaction Thermus thermophilus ethyl benzoylformate + NAD+
-
r
ethyl benzoylformate + NADH + H+ completely enantioselective reaction Thermus thermophilus ethyl (R)-mandelate + NAD+ 95% enantiomeric excess after 6 h at 50°C r
methyl (R)-mandelate + NADH + H+ 92% ethanoselective reaction Thermus thermophilus methyl benzoylformate + NAD+
-
r
methyl benzoylformate + NADH + H+ completely enantioselective reaction Thermus thermophilus methyl (R)-mandelate + NAD+ 92% enantiomeric excess after 6 h at 50°C r
additional information substrate specificity of ADHTt in the oxidation and the reduction reactions depends on the substrate, cf. (S)-1-phenylethanol dehydrogenase, detailed overview. The enzyme shows a high reduction rate with halogenated aryl ketones, such as 2,2,2-trifluoroacetophenone, 2-chloroacetophenone, and 4-chlorobutyrophenone, and with aryl diketones, such as 1-phenyl-1,2-propanedione, although it is not active on benzil, i.e. diphenylethanedione. ADHTt proves to be very effective in reducing aryl alpha-keto esters, although it is not active on aliphatic alpha-keto esters and aryl beta-keto ester. Critical role of the D37 residue in discriminating NAD(H) from NADP(H) Thermus thermophilus ?
-
?

Subunits

Subunits Comment Organism
tetramer 4 * 26961, recombinant enzyme, mass spectrometry, 4 * 26000, recombinant enzyme, SDS-PAGE Thermus thermophilus

Synonyms

Synonyms Comment Organism
ADHTt
-
Thermus thermophilus
More the enzyme belongs to the short-chain dehydrogenase/reductase, SDR, superfamily Thermus thermophilus
short-chain NAD(H)-dependent alcohol dehydrogenase
-
Thermus thermophilus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
60
-
oxidation reaction Thermus thermophilus
65
-
reduction reaction Thermus thermophilus

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
25 73 the enzyme displays activity at temperatures up to 73°C Thermus thermophilus

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
50
-
24 h, purified recombinant enzyme, 142% inactivation Thermus thermophilus
60
-
24 h, purified recombinant enzyme, 134% inactivation Thermus thermophilus
70
-
24 h, purified recombinant enzyme, 107% inactivation Thermus thermophilus
90
-
30 min, purified recombinant enzyme, 50% inactivation Thermus thermophilus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.84
-
NAD+ pH 6.0, 65°C, recombinant enzyme, reduction reaction Thermus thermophilus
25.5
-
2,2,2-trifluoroacetophenone pH 6.0, 65°C, recombinant enzyme, reduction reaction Thermus thermophilus
50.1
-
ethylbenzoylformate pH 6.0, 65°C, recombinant enzyme, reduction reaction Thermus thermophilus
52.4
-
NADH pH 6.0, 65°C, recombinant enzyme, reduction reaction Thermus thermophilus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
reduction reaction Thermus thermophilus
10
-
oxidation reaction Thermus thermophilus

pH Range

pH Minimum pH Maximum Comment Organism
6 7.7 reduction reaction, high activity Thermus thermophilus
9 10 oxidation reaction, high activity Thermus thermophilus

Cofactor

Cofactor Comment Organism Structure
additional information the enzyme shows no activity with NADP(H) Thermus thermophilus
NAD+ critical role of the D37 residue in discriminating NAD(H) from NADP(H) Thermus thermophilus
NADH critical role of the D37 residue in discriminating NAD(H) from NADP(H) Thermus thermophilus