BRENDA - Enzyme Database

Determinants of dual substrate specificity revealed by the crystal structure of homoisocitrate dehydrogenase from Thermus thermophilus in complex with homoisocitrate-Mg(2+)-NADH

Takahashi, K.; Tomita, T.; Kuzuyama, T.; Nishiyama, M.; Biochem. Biophys. Res. Commun. 478, 1688-1693 (2016)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.1.1.286
recombinant expression in Escherichia coli strain Escherichia coli BL21(DE3)CodonPlus-RIL
Thermus thermophilus
Crystallization (Commentary)
EC Number
Crystallization
Organism
1.1.1.286
purified enzyme TtHICDH in quaternary complex with homoisocitrate, NADH, and Mg2+, X-ray diffraction strczure determination and analysis at 2.5 A resolution, molecular replacement using the apoform of TtHICDH, PDB ID 1X0L, at a resolution of 2.5 A
Thermus thermophilus
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
1.1.1.286
(2S,3S)-thiahomoisocitrate
-
Thermus thermophilus
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.286
0.21
-
homoisocitrate
pH 8.0, 60°C, recombinant enzyme
Thermus thermophilus
1.1.1.286
0.29
-
isocitrate
pH 8.0, 60°C, recombinant enzyme
Thermus thermophilus
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.286
Mg2+
binding strutcure analysis
Thermus thermophilus
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.286
homoisocitrate + NAD+
Thermus thermophilus
-
2-oxoadipate + CO2 + NADH + H+
-
-
r
1.1.1.286
homoisocitrate + NAD+
Thermus thermophilus DSM 7039
-
2-oxoadipate + CO2 + NADH + H+
-
-
r
1.1.1.286
isocitrate + NAD+
Thermus thermophilus
-
2-oxoglutarate + CO2 + NADH
-
-
r
1.1.1.286
isocitrate + NAD+
Thermus thermophilus DSM 7039
-
2-oxoglutarate + CO2 + NADH
-
-
r
1.1.1.286
additional information
Thermus thermophilus
in contrast to other homoisicitrate dehydrogenases, the homoisocitrate dehydrogenase from the thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both homoisocitrate and isocitrate as substrates at similar efficiencies
?
-
-
-
1.1.1.286
additional information
Thermus thermophilus DSM 7039
in contrast to other homoisicitrate dehydrogenases, the homoisocitrate dehydrogenase from the thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both homoisocitrate and isocitrate as substrates at similar efficiencies
?
-
-
-
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.1.1.286
Thermus thermophilus
Q72IW9
-
-
1.1.1.286
Thermus thermophilus DSM 7039
Q72IW9
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.1.1.286
recombinant enzyme from Escherichia coli by anion exchange chromatography, ammonium sufate fractionation, hydrophobic interaction chromatography, and gel filtration
Thermus thermophilus
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.286
homoisocitrate + NAD+
-
740000
Thermus thermophilus
2-oxoadipate + CO2 + NADH + H+
-
-
-
r
1.1.1.286
homoisocitrate + NAD+
-
740000
Thermus thermophilus DSM 7039
2-oxoadipate + CO2 + NADH + H+
-
-
-
r
1.1.1.286
isocitrate + NAD+
-
740000
Thermus thermophilus
2-oxoglutarate + CO2 + NADH
-
-
-
r
1.1.1.286
isocitrate + NAD+
-
740000
Thermus thermophilus DSM 7039
2-oxoglutarate + CO2 + NADH
-
-
-
r
1.1.1.286
additional information
in contrast to other homoisicitrate dehydrogenases, the homoisocitrate dehydrogenase from the thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both homoisocitrate and isocitrate as substrates at similar efficiencies
740000
Thermus thermophilus
?
-
-
-
-
1.1.1.286
additional information
dual substrate specificity for homoisocitrate dehydrogenase
740000
Thermus thermophilus
?
-
-
-
-
1.1.1.286
additional information
in contrast to other homoisicitrate dehydrogenases, the homoisocitrate dehydrogenase from the thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both homoisocitrate and isocitrate as substrates at similar efficiencies
740000
Thermus thermophilus DSM 7039
?
-
-
-
-
1.1.1.286
additional information
dual substrate specificity for homoisocitrate dehydrogenase
740000
Thermus thermophilus DSM 7039
?
-
-
-
-
Subunits
EC Number
Subunits
Commentary
Organism
1.1.1.286
tetramer
dimer of dimers
Thermus thermophilus
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.286
60
-
assay at
Thermus thermophilus
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.286
33
-
homoisocitrate
pH 8.0, 60°C, recombinant enzyme
Thermus thermophilus
1.1.1.286
76
-
isocitrate
pH 8.0, 60°C, recombinant enzyme
Thermus thermophilus
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.286
8
-
assay at
Thermus thermophilus
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.286
NAD+
-
Thermus thermophilus
1.1.1.286
NADH
NADH forms specific contacts with enzyme TtHICDH. The 2'- and 3-OHs of the adenine ribose of NADH form hydrogen bonds with Asp265 conserved among HICDHs, which may serve as a determinant for the preference of HICDH family members for NAD+ to NADP+
Thermus thermophilus
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.286
recombinant expression in Escherichia coli strain Escherichia coli BL21(DE3)CodonPlus-RIL
Thermus thermophilus
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.286
NAD+
-
Thermus thermophilus
1.1.1.286
NADH
NADH forms specific contacts with enzyme TtHICDH. The 2'- and 3-OHs of the adenine ribose of NADH form hydrogen bonds with Asp265 conserved among HICDHs, which may serve as a determinant for the preference of HICDH family members for NAD+ to NADP+
Thermus thermophilus
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
1.1.1.286
purified enzyme TtHICDH in quaternary complex with homoisocitrate, NADH, and Mg2+, X-ray diffraction strczure determination and analysis at 2.5 A resolution, molecular replacement using the apoform of TtHICDH, PDB ID 1X0L, at a resolution of 2.5 A
Thermus thermophilus
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
1.1.1.286
(2S,3S)-thiahomoisocitrate
-
Thermus thermophilus
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.286
0.21
-
homoisocitrate
pH 8.0, 60°C, recombinant enzyme
Thermus thermophilus
1.1.1.286
0.29
-
isocitrate
pH 8.0, 60°C, recombinant enzyme
Thermus thermophilus
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.286
Mg2+
binding strutcure analysis
Thermus thermophilus
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.286
homoisocitrate + NAD+
Thermus thermophilus
-
2-oxoadipate + CO2 + NADH + H+
-
-
r
1.1.1.286
homoisocitrate + NAD+
Thermus thermophilus DSM 7039
-
2-oxoadipate + CO2 + NADH + H+
-
-
r
1.1.1.286
isocitrate + NAD+
Thermus thermophilus
-
2-oxoglutarate + CO2 + NADH
-
-
r
1.1.1.286
isocitrate + NAD+
Thermus thermophilus DSM 7039
-
2-oxoglutarate + CO2 + NADH
-
-
r
1.1.1.286
additional information
Thermus thermophilus
in contrast to other homoisicitrate dehydrogenases, the homoisocitrate dehydrogenase from the thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both homoisocitrate and isocitrate as substrates at similar efficiencies
?
-
-
-
1.1.1.286
additional information
Thermus thermophilus DSM 7039
in contrast to other homoisicitrate dehydrogenases, the homoisocitrate dehydrogenase from the thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both homoisocitrate and isocitrate as substrates at similar efficiencies
?
-
-
-
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.286
recombinant enzyme from Escherichia coli by anion exchange chromatography, ammonium sufate fractionation, hydrophobic interaction chromatography, and gel filtration
Thermus thermophilus
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.286
homoisocitrate + NAD+
-
740000
Thermus thermophilus
2-oxoadipate + CO2 + NADH + H+
-
-
-
r
1.1.1.286
homoisocitrate + NAD+
-
740000
Thermus thermophilus DSM 7039
2-oxoadipate + CO2 + NADH + H+
-
-
-
r
1.1.1.286
isocitrate + NAD+
-
740000
Thermus thermophilus
2-oxoglutarate + CO2 + NADH
-
-
-
r
1.1.1.286
isocitrate + NAD+
-
740000
Thermus thermophilus DSM 7039
2-oxoglutarate + CO2 + NADH
-
-
-
r
1.1.1.286
additional information
in contrast to other homoisicitrate dehydrogenases, the homoisocitrate dehydrogenase from the thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both homoisocitrate and isocitrate as substrates at similar efficiencies
740000
Thermus thermophilus
?
-
-
-
-
1.1.1.286
additional information
dual substrate specificity for homoisocitrate dehydrogenase
740000
Thermus thermophilus
?
-
-
-
-
1.1.1.286
additional information
in contrast to other homoisicitrate dehydrogenases, the homoisocitrate dehydrogenase from the thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both homoisocitrate and isocitrate as substrates at similar efficiencies
740000
Thermus thermophilus DSM 7039
?
-
-
-
-
1.1.1.286
additional information
dual substrate specificity for homoisocitrate dehydrogenase
740000
Thermus thermophilus DSM 7039
?
-
-
-
-
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.1.1.286
tetramer
dimer of dimers
Thermus thermophilus
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.286
60
-
assay at
Thermus thermophilus
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.286
33
-
homoisocitrate
pH 8.0, 60°C, recombinant enzyme
Thermus thermophilus
1.1.1.286
76
-
isocitrate
pH 8.0, 60°C, recombinant enzyme
Thermus thermophilus
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.286
8
-
assay at
Thermus thermophilus
General Information
EC Number
General Information
Commentary
Organism
1.1.1.286
evolution
homoisocitrate dehydrogenase, HICDH, is a member of the beta-decarboxylating dehydrogenase family
Thermus thermophilus
1.1.1.286
metabolism
in contrast to other homoisocitrate dehydrogenases, the enzyme from the thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both homoisocitrate and isocitrate as substrates at similar efficiencies. The enzyme catalyzes the conversion of homoisocitrate to 2-oxoadipate using NAD+ as a coenzyme, which is the fourth reaction involved in lysine biosynthesis through the alpha-aminoadipate pathway
Thermus thermophilus
1.1.1.286
additional information
enzyme structure modelling and molecular dynamics, the distal carboxyl group of homoiscitrate is recognized by the side chains of Ser72 and Arg85 from one subunit, and Asn173 from another subunit of a dimer unit. The enzyme recognizes the distal carboxyl group of isocitrate by Arg85 in the model. Active site structure analysis, the active site is located in the cleft between two domains. In the quaternary complex of TtHICDH, the basic residues, Arg88, Arg96, Arg118, Tyr125, and Lys171, recognize the malate moiety of HIC. Asp204 (from the otherdimer part) , Asp228, Asp232, and water molecules bind a Mg2+ ion in an octahedral coordination manner similar to those of other substrate-bound structures, e.g. PDB ID 4F7I
Thermus thermophilus
General Information (protein specific)
EC Number
General Information
Commentary
Organism
1.1.1.286
evolution
homoisocitrate dehydrogenase, HICDH, is a member of the beta-decarboxylating dehydrogenase family
Thermus thermophilus
1.1.1.286
metabolism
in contrast to other homoisocitrate dehydrogenases, the enzyme from the thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both homoisocitrate and isocitrate as substrates at similar efficiencies. The enzyme catalyzes the conversion of homoisocitrate to 2-oxoadipate using NAD+ as a coenzyme, which is the fourth reaction involved in lysine biosynthesis through the alpha-aminoadipate pathway
Thermus thermophilus
1.1.1.286
additional information
enzyme structure modelling and molecular dynamics, the distal carboxyl group of homoiscitrate is recognized by the side chains of Ser72 and Arg85 from one subunit, and Asn173 from another subunit of a dimer unit. The enzyme recognizes the distal carboxyl group of isocitrate by Arg85 in the model. Active site structure analysis, the active site is located in the cleft between two domains. In the quaternary complex of TtHICDH, the basic residues, Arg88, Arg96, Arg118, Tyr125, and Lys171, recognize the malate moiety of HIC. Asp204 (from the otherdimer part) , Asp228, Asp232, and water molecules bind a Mg2+ ion in an octahedral coordination manner similar to those of other substrate-bound structures, e.g. PDB ID 4F7I
Thermus thermophilus
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.1.1.286
157.1
-
homoisocitrate
pH 8.0, 60°C, recombinant enzyme
Thermus thermophilus
1.1.1.286
262.1
-
isocitrate
pH 8.0, 60°C, recombinant enzyme
Thermus thermophilus
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.1.1.286
157.1
-
homoisocitrate
pH 8.0, 60°C, recombinant enzyme
Thermus thermophilus
1.1.1.286
262.1
-
isocitrate
pH 8.0, 60°C, recombinant enzyme
Thermus thermophilus