Identification of catalytically important amino acid residues for enzymatic reduction of glyoxylate in plants
Hoover, G.J.; Jorgensen, R.; Rochon, A.; Bajwa, V.S.; Merrill, A.R.; Shelp, B.J.; Biochim. Biophys. Acta 1834, 2663-2671 (2013) 
Data extracted from this reference:
Cloned(Commentary)
Cloned (Commentary)
Organism
gene GLYR1, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 pLysS
Arabidopsis thaliana
Crystallization (Commentary)
Crystallization (Commentary)
Organism
purified apo-enzyme, sitting drop vapor diffusion method, mixing of 0.002 ml of protein solution with 0.002 ml of reservoir solution containing 0.2 M calcium acetate hydrate, 20% PEG 3350, pH 6.5, 20°C, 6 weeks, X-ray diffraction structure determination and analysis at 2.1 A resolution, molecular replacement using a previously unrecognized member of the beta-HAD family, cytokine-like nuclear factor, structure
Arabidopsis thaliana
Engineering
Protein Variants
Commentary
Organism
D239A
site-directed mutagenesis
Arabidopsis thaliana
F231A
site-directed mutagenesis
Arabidopsis thaliana
K170A
site-directed mutagenesis, catalytically inactive mutant
Arabidopsis thaliana
K170E
site-directed mutagenesis, the mutant shows highly reduced kcat for glyoxylate compared to the wild-type
Arabidopsis thaliana
K170H
site-directed mutagenesis, the mutant shows highly reduced kcat for glyoxylate compared to the wild-type
Arabidopsis thaliana
K170R
site-directed mutagenesis, the mutant shows highly reduced kcat for glyoxylate compared to the wild-type
Arabidopsis thaliana
N174A
site-directed mutagenesis
Arabidopsis thaliana
S121A
site-directed mutagenesis
Arabidopsis thaliana
T95A
site-directed mutagenesis
Arabidopsis thaliana
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
Michaelis-Menten kinetics, the activities of the mutant enzymes with succinic semialdehyde are generally too low for kinetic studies
Arabidopsis thaliana
0.0022
-
NADPH
pH 7.8, temperature not specified in the publication, recombinant wild-type enzyme, value determined with the use of a double beam spectrophotometer
Arabidopsis thaliana
0.87
-
Succinic semialdehyde
pH 7.8, temperature not specified in the publication, recombinant wild-type enzyme, value determined with the use of a double beam spectrophotometer
Arabidopsis thaliana
Localization
Localization
Commentary
Organism
GeneOntology No.
Textmining
cytosol
-
Arabidopsis thaliana
5829
-
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
additional information
Arabidopsis thaliana
the recombinant AtGLYR1 prefers NADPH over NADH and converts glyoxylate to glycolate, the enzyme has negligible hydroxypyruvate-dependent activity. Isozyme AtGLYR1 also converts succinic semialdehyde to gamma-hydroxybutyrate, albeit with much lower catalytic efficiency than for glyoxylate
?
-
-
?
succinic semialdehyde + NADPH + H+
Arabidopsis thaliana
-
4-hydroxybutyrate + NADP+
-
-
?
Organism
Organism
UniProt
Commentary
Textmining
Arabidopsis thaliana
Q9LSV0
-
-
Purification (Commentary)
Purification (Commentary)
Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 pLysS by precipitation with 10% PEG 8000, and nickel affinity chromatography
Arabidopsis thaliana
Reaction
Reaction
Commentary
Organism
Reaction ID
4-hydroxybutanoate + NADP+ = succinate semialdehyde + NADPH + H+
the enzyme performs an acid/base catalytic mechanism involving Lys170 as the general acid and a conserved active-site water molecule
Arabidopsis thaliana
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
additional information
the recombinant AtGLYR1 prefers NADPH over NADH and converts glyoxylate to glycolate, the enzyme has negligible hydroxypyruvate-dependent activity. Isozyme AtGLYR1 also converts succinic semialdehyde to gamma-hydroxybutyrate, albeit with much lower catalytic efficiency than for glyoxylate
737745
Arabidopsis thaliana
?
-
-
-
?
succinic semialdehyde + NADPH + H+
-
737745
Arabidopsis thaliana
4-hydroxybutyrate + NADP+
-
-
-
?
Subunits
Subunits
Commentary
Organism
More
enzyme domain structure analysis, overview
Arabidopsis thaliana
Synonyms
Synonyms
Commentary
Organism
At3g25530
-
Arabidopsis thaliana
AtGLYR1
-
Arabidopsis thaliana
SSA
-
Arabidopsis thaliana
succinic semialdehyde reductase
-
Arabidopsis thaliana
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.8
-
assay at
Arabidopsis thaliana
Cofactor
Cofactor
Commentary
Organism
Structure
additional information
recombinant AtGLYR1 prefers NADPH over NADH
Arabidopsis thaliana
NADP+
-
Arabidopsis thaliana
NADPH
-
Arabidopsis thaliana
Cloned(Commentary) (protein specific)
Commentary
Organism
gene GLYR1, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 pLysS
Arabidopsis thaliana
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
additional information
recombinant AtGLYR1 prefers NADPH over NADH
Arabidopsis thaliana
NADP+
-
Arabidopsis thaliana
NADPH
-
Arabidopsis thaliana
Crystallization (Commentary) (protein specific)
Crystallization
Organism
purified apo-enzyme, sitting drop vapor diffusion method, mixing of 0.002 ml of protein solution with 0.002 ml of reservoir solution containing 0.2 M calcium acetate hydrate, 20% PEG 3350, pH 6.5, 20°C, 6 weeks, X-ray diffraction structure determination and analysis at 2.1 A resolution, molecular replacement using a previously unrecognized member of the beta-HAD family, cytokine-like nuclear factor, structure
Arabidopsis thaliana
Engineering (protein specific)
Protein Variants
Commentary
Organism
D239A
site-directed mutagenesis
Arabidopsis thaliana
F231A
site-directed mutagenesis
Arabidopsis thaliana
K170A
site-directed mutagenesis, catalytically inactive mutant
Arabidopsis thaliana
K170E
site-directed mutagenesis, the mutant shows highly reduced kcat for glyoxylate compared to the wild-type
Arabidopsis thaliana
K170H
site-directed mutagenesis, the mutant shows highly reduced kcat for glyoxylate compared to the wild-type
Arabidopsis thaliana
K170R
site-directed mutagenesis, the mutant shows highly reduced kcat for glyoxylate compared to the wild-type
Arabidopsis thaliana
N174A
site-directed mutagenesis
Arabidopsis thaliana
S121A
site-directed mutagenesis
Arabidopsis thaliana
T95A
site-directed mutagenesis
Arabidopsis thaliana
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
Michaelis-Menten kinetics, the activities of the mutant enzymes with succinic semialdehyde are generally too low for kinetic studies
Arabidopsis thaliana
0.0022
-
NADPH
pH 7.8, temperature not specified in the publication, recombinant wild-type enzyme, value determined with the use of a double beam spectrophotometer
Arabidopsis thaliana
0.87
-
Succinic semialdehyde
pH 7.8, temperature not specified in the publication, recombinant wild-type enzyme, value determined with the use of a double beam spectrophotometer
Arabidopsis thaliana
Localization (protein specific)
Localization
Commentary
Organism
GeneOntology No.
Textmining
cytosol
-
Arabidopsis thaliana
5829
-
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
additional information
Arabidopsis thaliana
the recombinant AtGLYR1 prefers NADPH over NADH and converts glyoxylate to glycolate, the enzyme has negligible hydroxypyruvate-dependent activity. Isozyme AtGLYR1 also converts succinic semialdehyde to gamma-hydroxybutyrate, albeit with much lower catalytic efficiency than for glyoxylate
?
-
-
?
succinic semialdehyde + NADPH + H+
Arabidopsis thaliana
-
4-hydroxybutyrate + NADP+
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 pLysS by precipitation with 10% PEG 8000, and nickel affinity chromatography
Arabidopsis thaliana
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
additional information
the recombinant AtGLYR1 prefers NADPH over NADH and converts glyoxylate to glycolate, the enzyme has negligible hydroxypyruvate-dependent activity. Isozyme AtGLYR1 also converts succinic semialdehyde to gamma-hydroxybutyrate, albeit with much lower catalytic efficiency than for glyoxylate
737745
Arabidopsis thaliana
?
-
-
-
?
succinic semialdehyde + NADPH + H+
-
737745
Arabidopsis thaliana
4-hydroxybutyrate + NADP+
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
More
enzyme domain structure analysis, overview
Arabidopsis thaliana
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.8
-
assay at
Arabidopsis thaliana
General Information
General Information
Commentary
Organism
evolution
the primary sequence of cytosolic AtGLYR1 reveals several sequence elements that are consistent with the beta-HAD (beta-hydroxyacid dehydrogenase) protein family, sequence alignment of AtGLYR1 and beta-HAD family members, overview
Arabidopsis thaliana
additional information
identification of catalytically important amino acid residues for enzymatic reduction of glyoxylate in plants by bifunctional enzyme glyoxylate/succinic semialdehyde reductase 1, that converts both glyoxylate and succinic semialdehyde into their corresponding hydroxyacid equivalents. Residue Lys170 is essential for catalysis, Phe231, Asp239, Ser121 and Thr95 are more important in substrate binding than in catalysis, and Asn174 is more important in catalysis
Arabidopsis thaliana
General Information (protein specific)
General Information
Commentary
Organism
evolution
the primary sequence of cytosolic AtGLYR1 reveals several sequence elements that are consistent with the beta-HAD (beta-hydroxyacid dehydrogenase) protein family, sequence alignment of AtGLYR1 and beta-HAD family members, overview
Arabidopsis thaliana
additional information
identification of catalytically important amino acid residues for enzymatic reduction of glyoxylate in plants by bifunctional enzyme glyoxylate/succinic semialdehyde reductase 1, that converts both glyoxylate and succinic semialdehyde into their corresponding hydroxyacid equivalents. Residue Lys170 is essential for catalysis, Phe231, Asp239, Ser121 and Thr95 are more important in substrate binding than in catalysis, and Asn174 is more important in catalysis
Arabidopsis thaliana
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
11.6
-
Succinic semialdehyde
pH 7.8, temperature not specified in the publication, recombinant wild-type enzyme, value determined with the use of a double beam spectrophotometer
Arabidopsis thaliana
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
11.6
-
Succinic semialdehyde
pH 7.8, temperature not specified in the publication, recombinant wild-type enzyme, value determined with the use of a double beam spectrophotometer
Arabidopsis thaliana
Other publictions for EC 1.1.1.B47
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Synonyms
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
737745
Hoover
Identification of catalyticall ...
Arabidopsis thaliana
Biochim. Biophys. Acta
1834
2663-2671
2013
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1
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9
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1
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1
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4
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1
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1
9
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1
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1
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1
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2
2
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1
1
698628
Kockelkorn
Malonic semialdehyde reductase ...
Metallosphaera sedula, Metallosphaera sedula DSM 5348
J. Bacteriol.
191
6352-6362
2009
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1
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