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Literature summary for 1.1.1.86 extracted from

  • Chen, C.; Ko, T.; Lin, K.; Lin, B.; Huang, C.; Chiang, C.; Horng, J.
    NADH/NADPH bi-cofactor-utilizing and thermoactive ketol-acid reductoisomerase from Sulfolobus acidocaldarius (2018), Sci. Rep., 8, 7176 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.1.1.86

Application

Application Comment Organism
industry Sac-KARI is one of the rare enzymes that displays high activity at elevated temperatures, a broad range of pH tolerance and is able to utilize both NADH and NADPH as cofactors. These properties make it a potential candidate for use in metabolic engineering or industrial applications under anaerobic or harsh conditions Sulfolobus acidocaldarius

Cloned(Commentary)

Cloned (Comment) Organism
overexpression in Escherichia coli BL21 (DE3) Sulfolobus acidocaldarius

Crystallization (Commentary)

Crystallization (Comment) Organism
crystal structure at a 1.75 A resolution Sulfolobus acidocaldarius

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0004
-
 NADPH pH 8.0, 25°C Sulfolobus acidocaldarius
0.006
-
 NADH pH 8.0, 25°C Sulfolobus acidocaldarius
0.06
-
 (S)-2-acetolactate pH 8.0, 60°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.074
-
 (S)-2-acetolactate pH 8.0, 37°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.091
-
 (S)-2-acetolactate pH 8.0, 60°C, 6 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.093
-
 (S)-2-acetolactate pH 8.0, 60°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.099
-
 (S)-2-acetolactate pH 8.0, 37°C, 10 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.14
-
 (S)-2-acetolactate pH 8.0, 25°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.144
-
 (S)-2-acetolactate pH 8.0, 60°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.157
-
 (S)-2-acetolactate pH 8.0, 37°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.165
-
 (S)-2-acetolactate pH 8.0, 37°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.184
-
 (S)-2-acetolactate pH 8.0, 25°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.188
-
 (S)-2-acetolactate pH 8.0, 25°C, 10 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.469
-
 (S)-2-acetolactate pH 8.0, 25°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ the enzyme binds two Mg2+ ions in the active site to bridge the substrate ligand and protein. The substrate binding and catalytic efficiency of KARI are highly dependent on the Mg2+ concentration Sulfolobus acidocaldarius

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
70000
-
 gel filtration Sulfolobus acidocaldarius

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
(S)-2-acetolactate + NADPH + H+ Sulfolobus acidocaldarius
-
 (2R)-2,3-dihydroxy-3-methylbutanoate + NADP+
-
 ?
(S)-2-acetolactate + NADPH + H+ Sulfolobus acidocaldarius ATCC 33909
-
 (2R)-2,3-dihydroxy-3-methylbutanoate + NADP+
-
 ?

Organism

Organism UniProt Comment Textmining
Sulfolobus acidocaldarius Q4J8K9
-
-
Sulfolobus acidocaldarius ATCC 33909 Q4J8K9
-
-

Purification (Commentary)

Purification (Comment) Organism
-
 Sulfolobus acidocaldarius

Renatured (Commentary)

Renatured (Comment) Organism
at 95°C, the enzyme is completely unfolded, and it does not refold even when it is slowly cooled to 25°C. Presumably the unfolding process is irreversible, especially after dissociation of the intertwined knot domains Sulfolobus acidocaldarius

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(S)-2-acetolactate + NADH + H+ the enzyme can also use NADH for efficient catalysis. Higher activity by with NADPH as compared to NADH. kcat/Km ratio of NADH/NADPH is 0.051 Sulfolobus acidocaldarius (2R)-2,3-dihydroxy-3-methylbutanoate + NAD+
-
 ?
(S)-2-acetolactate + NADH + H+ the enzyme can also use NADH for efficient catalysis. Higher activity by with NADPH as compared to NADH. kcat/Km ratio of NADH/NADPH is 0.051 Sulfolobus acidocaldarius ATCC 33909 (2R)-2,3-dihydroxy-3-methylbutanoate + NAD+
-
 ?
(S)-2-acetolactate + NADPH + H+
-
 Sulfolobus acidocaldarius (2R)-2,3-dihydroxy-3-methylbutanoate + NADP+
-
 ?
(S)-2-acetolactate + NADPH + H+ the enzyme can also use NADH for efficient catalysis. Higher activity by with NADPH as compared to NADH. kcat/Km ratio of NADH/NADPH is 0.051 Sulfolobus acidocaldarius (2R)-2,3-dihydroxy-3-methylbutanoate + NADP+
-
 ?
(S)-2-acetolactate + NADPH + H+
-
 Sulfolobus acidocaldarius ATCC 33909 (2R)-2,3-dihydroxy-3-methylbutanoate + NADP+
-
 ?
(S)-2-acetolactate + NADPH + H+ the enzyme can also use NADH for efficient catalysis. Higher activity by with NADPH as compared to NADH. kcat/Km ratio of NADH/NADPH is 0.051 Sulfolobus acidocaldarius ATCC 33909 (2R)-2,3-dihydroxy-3-methylbutanoate + NADP+
-
 ?

Subunits

Subunits Comment Organism
homodimer
-
 Sulfolobus acidocaldarius

Synonyms

Synonyms Comment Organism
KARI
-
 Sulfolobus acidocaldarius

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
60
-
 the secondary structure of the enzyme remains intact in the presence of 2 mM Mg2+ at 60°C Sulfolobus acidocaldarius
65 95 the enzyme retains most of its native folding, when the temperature is increased to 65°C. At 95°C, the enzyme is completely unfolded Sulfolobus acidocaldarius

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
3 8 the enzyme exhibits pH-tolerance over a broader range (pH 3.0-8.0), which is preferred in industrial application Sulfolobus acidocaldarius

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
86
-
 Tm-value Sulfolobus acidocaldarius
95
-
 at 95°C, the enzyme is completely unfolded, and it does not refold even when it is slowly cooled to 25°C. Presumably the unfolding process is irreversible, especially after dissociation of the intertwined knot domains Sulfolobus acidocaldarius

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.055
-
 (S)-2-acetolactate pH 8.0, 25°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.056
-
 NADH pH 8.0, 25°C Sulfolobus acidocaldarius
0.058
-
 (S)-2-acetolactate pH 8.0, 25°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.06
-
 (S)-2-acetolactate pH 8.0, 25°C, 10 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.064
-
 NADPH pH 8.0, 25°C Sulfolobus acidocaldarius
0.068
-
 (S)-2-acetolactate pH 8.0, 25°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.157
-
 (S)-2-acetolactate pH 8.0, 37°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.164
-
 (S)-2-acetolactate pH 8.0, 37°C, 10 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.192
-
 (S)-2-acetolactate pH 8.0, 37°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.195
-
 (S)-2-acetolactate pH 8.0, 37°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.444
-
 (S)-2-acetolactate pH 8.0, 60°C, 6 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.526
-
 (S)-2-acetolactate pH 8.0, 60°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.876
-
 (S)-2-acetolactate pH 8.0, 60°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.957
-
 (S)-2-acetolactate pH 8.0, 60°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius

Cofactor

Cofactor Comment Organism Structure
NADH higher activity by with NADPH as compared to NADH. kcat/Km ratio of NADH/NADPH is 0.051 Sulfolobus acidocaldarius
NADPH higher activity by with NADPH as compared to NADH. kcat/Km ratio of NADH/NADPH is 0.051 Sulfolobus acidocaldarius

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.12
-
 (S)-2-acetolactate pH 8.0, 25°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.32
-
 (S)-2-acetolactate pH 8.0, 25°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.33
-
 (S)-2-acetolactate pH 8.0, 25°C, 10 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
0.49
-
 (S)-2-acetolactate pH 8.0, 25°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
0.95
-
 (S)-2-acetolactate pH 8.0, 37°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.23
-
 (S)-2-acetolactate pH 8.0, 37°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.66
-
 (S)-2-acetolactate pH 8.0, 37°C, 10 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
2.63
-
 (S)-2-acetolactate pH 8.0, 37°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
3.66
-
 (S)-2-acetolactate pH 8.0, 60°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
4.88
-
 (S)-2-acetolactate pH 8.0, 60°C, 6 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
9.5
-
 NADH pH 8.0, 25°C Sulfolobus acidocaldarius
10.3
-
 (S)-2-acetolactate pH 8.0, 60°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
14.58
-
 (S)-2-acetolactate pH 8.0, 60°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
185.8
-
 NADPH pH 8.0, 25°C Sulfolobus acidocaldarius