Cloned (Comment) | Organism |
---|---|
gene GLYR1, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 pLysS | Arabidopsis thaliana |
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 | 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 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 | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
cytosol | - |
Arabidopsis thaliana | 5829 | - |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Arabidopsis thaliana | Q9LSV0 | - |
- |
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 | 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 | 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 | Comment | Organism |
---|---|---|
More | enzyme domain structure analysis, overview | Arabidopsis thaliana |
Synonyms | Comment | Organism |
---|---|---|
At3g25530 | - |
Arabidopsis thaliana |
AtGLYR1 | - |
Arabidopsis thaliana |
SSA | - |
Arabidopsis thaliana |
succinic semialdehyde reductase | - |
Arabidopsis thaliana |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.8 | - |
assay at | Arabidopsis thaliana |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | recombinant AtGLYR1 prefers NADPH over NADH | Arabidopsis thaliana | |
NADP+ | - |
Arabidopsis thaliana | |
NADPH | - |
Arabidopsis thaliana |
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 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 |