Cloned (Comment) | Organism |
---|---|
gene GLYR1, sequence comparisons of GLYR genes and HPR genes | Arabidopsis thaliana |
gene GLYR1, sequence comparisons of GLYR genes and HPR genes, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 pLysS | Arabidopsis thaliana |
gene GLYR2, sequence comparisons of GLYR genes and HPR genes, recombinant expression of a His6-tagged truncated AtGLYR2 cDNA sequence, lacking the N-terminal 58 amino acids, 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 | Arabidopsis thaliana | |
0.0009 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant N174A | Arabidopsis thaliana | |
0.0018 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant S121A | Arabidopsis thaliana | |
0.002 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant F231A | Arabidopsis thaliana | |
0.0022 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a double beam spectrophotometer | Arabidopsis thaliana | |
0.0027 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant D239A | Arabidopsis thaliana | |
0.0034 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant wild-type enzyme | Arabidopsis thaliana | |
0.0045 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a double beam spectrophotometer | Arabidopsis thaliana | |
0.016 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, recombinant truncated enzyme | Arabidopsis thaliana | |
0.018 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant wild-type enzyme | Arabidopsis thaliana | |
0.033 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant K170E | Arabidopsis thaliana | |
0.061 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant K170R | Arabidopsis thaliana | |
0.0648 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant T95A | Arabidopsis thaliana | |
0.088 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant N174A | Arabidopsis thaliana | |
0.181 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant S121A | Arabidopsis thaliana | |
3 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant D239A | Arabidopsis thaliana | |
4.6 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant T95A | Arabidopsis thaliana | |
12.4 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant F231A | Arabidopsis thaliana |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
chloroplast | - |
Arabidopsis thaliana | 9507 | - |
cytosol | - |
Arabidopsis thaliana | 5829 | - |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
glycolate + NADP+ | Arabidopsis thaliana | - |
glyoxylate + NADPH + H+ | - |
r | |
additional information | Arabidopsis thaliana | HPR3 prefers NADPH over NADH and converts glyoxylate to glycolate, the purified recombinant HPR3 shows similar activity with hydroxypyruvate and glyoxylate | ? | - |
? | |
additional information | Arabidopsis thaliana | the recombinant AtGLYR1 prefers NADPH over NADH and converts glyoxylate to glycolate, AtGLYR1 has negligible hydroxypyruvate-dependent activity. Isozyme AtGLYR1 also converts succinic semialdehyde to gamma-hydroxybutyrate, albeit with much lower catalytic efficiency than for glyoxylate | ? | - |
? | |
additional information | Arabidopsis thaliana | the recombinant AtGLYR2 prefers NADPH over NADH and converts glyoxylate to glycolate, AtGLYR2 has negligible hydroxypyruvate-dependent activity. Isozyme AtGLYR2 also converts succinic semialdehyde to gamma-hydroxybutyrate, albeit with much lower catalytic efficiency than for glyoxylate | ? | - |
? | |
additional information | Arabidopsis thaliana | the recombinant AtHPR2 prefers NADPH over NADH but utilizes hydroxypyruvate and glyoxylate similarly | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Arabidopsis thaliana | A0A178WMD4 | - |
- |
Arabidopsis thaliana | F4I907 | - |
- |
Arabidopsis thaliana | Q9CA90 | - |
- |
Arabidopsis thaliana | Q9LSV0 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged truncated mutant enzyme from Escherichia coli strain BL21 pLysS by precipitation with 10% PEG 8000, and nickel affinity chromatography | Arabidopsis thaliana |
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 |
---|---|---|---|
glycolate + NADP+ = glyoxylate + 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. |
---|---|---|---|---|---|---|
glycolate + NADP+ | - |
Arabidopsis thaliana | glyoxylate + NADPH + H+ | - |
r | |
glyoxylate + NADPH + H+ | - |
Arabidopsis thaliana | glycolate + NADP+ | - |
r | |
additional information | HPR3 prefers NADPH over NADH and converts glyoxylate to glycolate, the purified recombinant HPR3 shows similar activity with hydroxypyruvate and glyoxylate | Arabidopsis thaliana | ? | - |
? | |
additional information | the recombinant AtGLYR1 prefers NADPH over NADH and converts glyoxylate to glycolate, AtGLYR1 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 | ? | - |
? | |
additional information | the recombinant AtGLYR2 prefers NADPH over NADH and converts glyoxylate to glycolate, AtGLYR2 has negligible hydroxypyruvate-dependent activity. Isozyme AtGLYR2 also converts succinic semialdehyde to gamma-hydroxybutyrate, albeit with much lower catalytic efficiency than for glyoxylate | Arabidopsis thaliana | ? | - |
? | |
additional information | the recombinant AtHPR2 prefers NADPH over NADH but utilizes hydroxypyruvate and glyoxylate similarly | Arabidopsis thaliana | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | domain I, with the dinucleotide binding region, comprises residues 1-165 in the N-terminus. This typical Rossmann fold domain contains two alpha/beta units: a six-stranded parallel beta-sheet (beta1-beta6a) covered by four helices (alpha1-alpha5) and followed by a mixed three-stranded beta-sheet (beta6b-beta8) covered by two helices (alpha6 and alpha7). Domain II (residues 195-287) consists of only helices (alpha8-alpha13) from the C-terminal segment of the protein. The two domains are connected by a long alpha-helix, alpha8 (residues 166-194). Enzyme domain structure analysis, overview | Arabidopsis thaliana |
Synonyms | Comment | Organism |
---|---|---|
At3g25530 | - |
Arabidopsis thaliana |
AtGLYR1 | - |
Arabidopsis thaliana |
AtGLYR2 | - |
Arabidopsis thaliana |
glycerate dehydrogenase | - |
Arabidopsis thaliana |
GLYR1 | - |
Arabidopsis thaliana |
GLYR2 | - |
Arabidopsis thaliana |
HPR2 | - |
Arabidopsis thaliana |
HPR3 | - |
Arabidopsis thaliana |
More | cf. EC 1.1.1.26 | Arabidopsis thaliana |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0052 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant K170E | Arabidopsis thaliana | |
0.051 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant K170R | Arabidopsis thaliana | |
6.06 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant N174A | Arabidopsis thaliana | |
9.09 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant N174A | Arabidopsis thaliana | |
9.56 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant F231A | Arabidopsis thaliana | |
11 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant F231A | Arabidopsis thaliana | |
22 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant D239A | Arabidopsis thaliana | |
25.4 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant D239A | Arabidopsis thaliana | |
51.1 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant T95A | Arabidopsis thaliana | |
54.6 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant wild-type enzyme | Arabidopsis thaliana | |
67.8 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant T95A | Arabidopsis thaliana | |
84.1 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant wild-type enzyme | Arabidopsis thaliana | |
86.4 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant S121A | Arabidopsis thaliana | |
93.7 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant S121A | Arabidopsis thaliana | |
3407 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant wild-type enzyme | 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 | |
additional information | recombinant AtGLYR2 prefers NADPH over NADH | Arabidopsis thaliana | |
additional information | recombinant HPR3 prefers NADPH over NADH | Arabidopsis thaliana | |
additional information | the recombinant AtHPR2 prefers NADPH over NADH | Arabidopsis thaliana | |
NADP+ | - |
Arabidopsis thaliana | |
NADPH | - |
Arabidopsis thaliana |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the beta-HAD (beta-hydroxyacid dehydrogenase) protein family | Arabidopsis thaliana |
evolution | the enzyme belongs to the beta-HAD (beta-hydroxyacid dehydrogenase) protein family. AtHPR2 and AtHPR3 are 45% identical to each other at the amino acid level, but only 19-25% identical to AtHPR1, the NADH-dependent form, and 8-9% identical to the AtGLYRs. None of the AtHPRs contains the active-site residues conserved in AtGLYR1 and AtGLYR2, indicating that the sites responsible for reducing glyoxylate differ greatly between the AtGLYRs and AtHPRs | Arabidopsis thaliana |
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. AtHPR2 and AtHPR3 are 45% identical to each other at the amino acid level, but only 19-25% identical to AtHPR1, the NADH-dependent form, and 8-9% identical to the AtGLYRs. None of the AtHPRs contains the active-site residues conserved in AtGLYR1 and AtGLYR2, indicating that the sites responsible for reducing glyoxylate differ greatly between the AtGLYRs and AtHPRs | Arabidopsis thaliana |
evolution | the primary sequence of plastidial AtGLYR2 reveals several sequence elements that are consistent with the beta-HAD (beta-hydroxyacid dehydrogenase) protein family, sequence alignment of AtGLYR2 and beta-HAD family members, overview. AtHPR2 and AtHPR3 are 45% identical to each other at the amino acid level, but only 19-25% identical to AtHPR1, the NADH-dependent form, and 8-9% identical to the AtGLYRs. None of the AtHPRs contains the active-site residues conserved in AtGLYR1 and AtGLYR2, indicating that the sites responsible for reducing glyoxylate differ greatly between the AtGLYRs and AtHPRs | 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. Residues Thr95, Phe231 and Asp239 serve a more important role in substrate orientation and docking than in catalysis | Arabidopsis thaliana |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.19 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant K170E | Arabidopsis thaliana | |
0.86 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant K170R | Arabidopsis thaliana | |
0.87 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant F231A | Arabidopsis thaliana | |
7.45 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant D239A | Arabidopsis thaliana | |
14.6 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant T95A | Arabidopsis thaliana | |
72.8 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant N174A | Arabidopsis thaliana | |
480 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant S121A | Arabidopsis thaliana | |
779 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant T95A | Arabidopsis thaliana | |
2870 | - |
glyoxylate | 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 | |
3407 | - |
glyoxylate | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant wild-type enzyme | Arabidopsis thaliana | |
4340 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant F231A | Arabidopsis thaliana | |
10400 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant D239A | Arabidopsis thaliana | |
10900 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant N174A | Arabidopsis thaliana | |
24450 | - |
NADPH | pH 7.8, temperature not specified in the publication, recombinant wild-type enzyme, value determined with the use of a microplate reader | Arabidopsis thaliana | |
51700 | - |
NADPH | pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant S121A | Arabidopsis thaliana |