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Literature summary for 1.1.1.B47 extracted from

  • Hoover, G.J.; Jorgensen, R.; Rochon, A.; Bajwa, V.S.; Merrill, A.R.; Shelp, B.J.
    Identification of catalytically important amino acid residues for enzymatic reduction of glyoxylate in plants (2013), Biochim. Biophys. Acta, 1834, 2663-2671.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene GLYR1, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 pLysS Arabidopsis thaliana

Crystallization (Commentary)

Crystallization (Comment) 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

Protein Variants

Protein Variants Comment 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 Comment 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 Comment Organism GeneOntology No. Textmining
cytosol
-
Arabidopsis thaliana 5829
-

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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 Comment Textmining
Arabidopsis thaliana Q9LSV0
-
-

Purification (Commentary)

Purification (Comment) 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 Comment 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 Comment Substrates Organism Products Comment (Products) Rev. Reac.
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 Arabidopsis thaliana ?
-
?
succinic semialdehyde + NADPH + H+
-
Arabidopsis thaliana 4-hydroxybutyrate + NADP+
-
?

Subunits

Subunits Comment Organism
More enzyme domain structure analysis, overview Arabidopsis thaliana

Synonyms

Synonyms Comment Organism
At3g25530
-
Arabidopsis thaliana
AtGLYR1
-
Arabidopsis thaliana
SSA
-
Arabidopsis thaliana
succinic semialdehyde reductase
-
Arabidopsis thaliana

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.8
-
assay at Arabidopsis thaliana

Cofactor

Cofactor Comment Organism Structure
additional information recombinant AtGLYR1 prefers NADPH over NADH Arabidopsis thaliana
NADP+
-
Arabidopsis thaliana
NADPH
-
Arabidopsis thaliana

General Information

General Information Comment 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 Comment 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