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Literature summary 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

Application

EC Number Application Comment Organism
1.1.1.86 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)

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

Crystallization (Commentary)

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

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.86 0.0004
-
 NADPH pH 8.0, 25°C Sulfolobus acidocaldarius
1.1.1.86 0.006
-
 NADH pH 8.0, 25°C Sulfolobus acidocaldarius
1.1.1.86 0.06
-
 (S)-2-acetolactate pH 8.0, 60°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.074
-
 (S)-2-acetolactate pH 8.0, 37°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.091
-
 (S)-2-acetolactate pH 8.0, 60°C, 6 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.093
-
 (S)-2-acetolactate pH 8.0, 60°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.099
-
 (S)-2-acetolactate pH 8.0, 37°C, 10 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.14
-
 (S)-2-acetolactate pH 8.0, 25°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.144
-
 (S)-2-acetolactate pH 8.0, 60°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.157
-
 (S)-2-acetolactate pH 8.0, 37°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.165
-
 (S)-2-acetolactate pH 8.0, 37°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.184
-
 (S)-2-acetolactate pH 8.0, 25°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.188
-
 (S)-2-acetolactate pH 8.0, 25°C, 10 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.469
-
 (S)-2-acetolactate pH 8.0, 25°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.1.1.86 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]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.1.1.86 70000
-
 gel filtration Sulfolobus acidocaldarius

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.86 Sulfolobus acidocaldarius Q4J8K9
-
-
1.1.1.86 Sulfolobus acidocaldarius ATCC 33909 Q4J8K9
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.86
-
 Sulfolobus acidocaldarius

Renatured (Commentary)

EC Number Renatured (Comment) Organism
1.1.1.86 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.86 (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+
-
 ?
1.1.1.86 (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+
-
 ?
1.1.1.86 (S)-2-acetolactate + NADPH + H+
-
 Sulfolobus acidocaldarius (2R)-2,3-dihydroxy-3-methylbutanoate + NADP+
-
 ?
1.1.1.86 (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+
-
 ?
1.1.1.86 (S)-2-acetolactate + NADPH + H+
-
 Sulfolobus acidocaldarius ATCC 33909 (2R)-2,3-dihydroxy-3-methylbutanoate + NADP+
-
 ?
1.1.1.86 (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

EC Number Subunits Comment Organism
1.1.1.86 homodimer
-
 Sulfolobus acidocaldarius

Synonyms

EC Number Synonyms Comment Organism
1.1.1.86 KARI
-
 Sulfolobus acidocaldarius

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.86 60
-
 the secondary structure of the enzyme remains intact in the presence of 2 mM Mg2+ at 60°C Sulfolobus acidocaldarius
1.1.1.86 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]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
1.1.1.86 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]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.1.1.86 86
-
 Tm-value Sulfolobus acidocaldarius
1.1.1.86 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]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.86 0.055
-
 (S)-2-acetolactate pH 8.0, 25°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.056
-
 NADH pH 8.0, 25°C Sulfolobus acidocaldarius
1.1.1.86 0.058
-
 (S)-2-acetolactate pH 8.0, 25°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.06
-
 (S)-2-acetolactate pH 8.0, 25°C, 10 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.064
-
 NADPH pH 8.0, 25°C Sulfolobus acidocaldarius
1.1.1.86 0.068
-
 (S)-2-acetolactate pH 8.0, 25°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.157
-
 (S)-2-acetolactate pH 8.0, 37°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.164
-
 (S)-2-acetolactate pH 8.0, 37°C, 10 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.192
-
 (S)-2-acetolactate pH 8.0, 37°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.195
-
 (S)-2-acetolactate pH 8.0, 37°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.444
-
 (S)-2-acetolactate pH 8.0, 60°C, 6 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.526
-
 (S)-2-acetolactate pH 8.0, 60°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.876
-
 (S)-2-acetolactate pH 8.0, 60°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.957
-
 (S)-2-acetolactate pH 8.0, 60°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.86 NADH higher activity by with NADPH as compared to NADH. kcat/Km ratio of NADH/NADPH is 0.051 Sulfolobus acidocaldarius
1.1.1.86 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.86 0.12
-
 (S)-2-acetolactate pH 8.0, 25°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.32
-
 (S)-2-acetolactate pH 8.0, 25°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.33
-
 (S)-2-acetolactate pH 8.0, 25°C, 10 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 0.49
-
 (S)-2-acetolactate pH 8.0, 25°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 0.95
-
 (S)-2-acetolactate pH 8.0, 37°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 1.23
-
 (S)-2-acetolactate pH 8.0, 37°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 1.66
-
 (S)-2-acetolactate pH 8.0, 37°C, 10 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 2.63
-
 (S)-2-acetolactate pH 8.0, 37°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 3.66
-
 (S)-2-acetolactate pH 8.0, 60°C, 2 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 4.88
-
 (S)-2-acetolactate pH 8.0, 60°C, 6 mM Mg2+, cofactor: NADH Sulfolobus acidocaldarius
1.1.1.86 9.5
-
 NADH pH 8.0, 25°C Sulfolobus acidocaldarius
1.1.1.86 10.3
-
 (S)-2-acetolactate pH 8.0, 60°C, 2 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 14.58
-
 (S)-2-acetolactate pH 8.0, 60°C, 10 mM Mg2+, cofactor: NADPH Sulfolobus acidocaldarius
1.1.1.86 185.8
-
 NADPH pH 8.0, 25°C Sulfolobus acidocaldarius