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Literature summary for 1.1.1.85 extracted from

  • Furukawa, R.; Toma, W.; Yamazaki, K.; Akanuma, S.
    Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties (2020), Sci. Rep., 10, 15493 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.1.1.85

Cloned(Commentary)

Cloned (Comment) Organism
two ancestral sequences of 3-isopropylmalate dehydrogenase (ancIPMDH-IQ and ancIPMDH-ML) are designed by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences are artificially synthesized and expressed in Escherichia coli synthetic construct

Protein Variants

Protein Variants Comment Organism
additional information two ancestral sequences of 3-isopropylmalate dehydrogenase are designed by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences are artificially synthesized and expressed in Escherichia coli. The ancestral sequence reconstruction can produce a thermally stable enzyme with catalytic properties adapted to low-temperature reactions synthetic construct

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0047
-
 (2R,3S)-3-isopropylmalate acIPMDH-ML, pH 8.0, 40°C synthetic construct
0.0053
-
 (2R,3S)-3-isopropylmalate ancIPMDH-IQ, pH 8.0, 40°C synthetic construct
0.01
-
 (2R,3S)-3-isopropylmalate acIPMDH-ML, pH 8.0, 70°C synthetic construct
0.01
-
 (2R,3S)-3-isopropylmalate ancIPMDH-IQ, pH 8.0, 70°C synthetic construct
0.037
-
 NAD+ acIPMDH-ML, pH 8.0, 25°C synthetic construct
0.045
-
 NAD+ ancIPMDH-IQ, pH 8.0, 25°C synthetic construct
0.054
-
 NAD+ acIPMDH-ML, pH 8.0, 40°C synthetic construct
0.067
-
 NAD+ ancIPMDH-IQ, pH 8.0, 40°C synthetic construct
0.73
-
 NAD+ acIPMDH-ML, pH 8.0, 70°C synthetic construct
0.96
-
 NAD+ ancIPMDH-IQ, pH 8.0, 70°C synthetic construct

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
(2R,3S)-3-isopropylmalate + NAD+ synthetic construct
-
 4-methyl-2-oxopentanoate + CO2 + NADH + H+
-
 ir

Organism

Organism UniProt Comment Textmining
synthetic construct
-
 two ancestral sequences of 3-isopropylmalate dehydrogenase (ancIPMDH-IQ and ancIPMDH-ML) are designed by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences are artificially synthesized and expressed in Escherichia coli. They have considerably higher low-temperature catalytic activity as compared with the Thermus thermophilus enzyme
-

Purification (Commentary)

Purification (Comment) Organism
-
 synthetic construct

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 the specific activities of ancIPMDH-IQ and ancIPMDHML are, respectively, 3.1fold and 2.1fold greater than that of Thermus thermophilus IPMDH at 25°C. At 70°C, the specificity activities of the ancestral IPMDHs are greater than those of the mesophilic IPMDHs but smaller than that of the thermophilic enzyme synthetic construct
1.87
-
 acIPMDH-ML, pH 8.0, 25°C synthetic construct
2.76
-
 ancIPMDH-IQ, pH 8.0, 25°C synthetic construct
38.5
-
 ancIPMDH-IQ, pH 8.0, 70°C synthetic construct
39.1
-
 acIPMDH-ML, pH 8.0, 70°C synthetic construct

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(2R,3S)-3-isopropylmalate + NAD+
-
 synthetic construct 4-methyl-2-oxopentanoate + CO2 + NADH + H+
-
 ir

Subunits

Subunits Comment Organism
dimer ancIPMDH-IQ synthetic construct
dimer predominantly a dimer, but a small elution peak corresponding to a tetramer is observed synthetic construct

Synonyms

Synonyms Comment Organism
IPMDH
-
 synthetic construct

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
75
-
 ancIPMDH-IQ synthetic construct
77
-
 acIPMDH-ML synthetic construct

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
25 50 although the ancestral IPMDHs (ancIPMDH-IQ and ancIPMDH-ML) are slightly less catalytically active relative to the mesophilic IPMDHs, they are more active than the thermophilic IPMDH at temperatures ranging from 25 to 50°C. ancIPMDH-IQ and ancIPMDH-ML functioned optimally at 70-80°C and maintain, respectively, 7% and 4% of their maximum activities at 25°C synthetic construct

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
 two ancestral sequences of 3-isopropylmalate dehydrogenase (ancIPMDH-IQ and ancIPMDH-ML) are designed by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus synthetic construct
88
-
 Tm-value, ancIPMDH-IQ synthetic construct
90
-
 Tm-value, ancIPMDH-ML synthetic construct

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.2
-
 NAD+ acIPMDH-ML, pH 8.0, 25°C synthetic construct
1.2
-
 (2R,3S)-3-isopropylmalate acIPMDH-ML, pH 8.0, 25°C synthetic construct
1.7
-
 NAD+ ancIPMDH-IQ, pH 8.0, 25°C synthetic construct
1.7
-
 (2R,3S)-3-isopropylmalate ancIPMDH-IQ, pH 8.0, 25°C synthetic construct
4.3
-
 NAD+ acIPMDH-ML, pH 8.0, 40°C synthetic construct
4.3
-
 (2R,3S)-3-isopropylmalate acIPMDH-ML, pH 8.0, 40°C synthetic construct
4.6
-
 NAD+ ancIPMDH-IQ, pH 8.0, 40°C synthetic construct
4.6
-
 (2R,3S)-3-isopropylmalate ancIPMDH-IQ, pH 8.0, 40°C synthetic construct
22
-
 NAD+ acIPMDH-ML, pH 8.0, 70°C synthetic construct
22
-
 (2R,3S)-3-isopropylmalate acIPMDH-ML, pH 8.0, 70°C synthetic construct
22
-
 NAD+ ancIPMDH-IQ, pH 8.0, 70°C synthetic construct
22
-
 (2R,3S)-3-isopropylmalate ancIPMDH-IQ, pH 8.0, 70°C synthetic construct