Cloned (Comment) | Organism |
---|---|
amplified from seedling cDNA library to encode 11-amino acid autoprocessing propeptide, N-terminal cysteine for an active enzyme and deleted chloroplast transit peptide, in pCR2.1TOPO for sequencing, in pET26b for expression in Escherichia coli BL21(DE3) | Arabidopsis thaliana |
Crystallization (Comment) | Organism |
---|---|
when mapped onto the structure from Bacillus subtilis GPRAT (PDB: 1AO0), the site of mutation R264K lies within 10 A of the glutaminase site | Arabidopsis thaliana |
Protein Variants | Comment | Organism |
---|---|---|
R264K | increased resistance to DAS734 with >500-fold higher IC50 compared to wild-type: no inhibition by 100 microM DAS734 and no effect on reaction time course, the same allosteric inhibition by adenine nucleotides as the wild-type, Arg-264 conserved in almost every GPRAT sequence | Arabidopsis thaliana |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
adenine nucleotide | maximal inhibition by combination of adenine nucleotides (in total 10 mM), inhibitory effect of single nucleotides (5 mM) increases in the order AMP, ATP, ADP | Arabidopsis thaliana | |
DAS734 | phenyltriazole acetic acid herbicide, [5-(4-chlorophenyl)-1-isopropyl-1H-[1,2,4]triazol-3-yl]-acetic acid, inhibits Arabidopsis root growth by 50% at 200 nM, phytotoxicity can be alleviated by addition of adenine, no inhibitory effect on Escherichia coli; phenyltriazole acetic acid herbicide, [5-(4-chlorophenyl)-1-isopropyl-1H-[1,2,4]triazol-3-yl]-acetic acid, non-competitive with respect to L-glutamine with slow, tight-binding behavior, inhibits Arabidopsis root growth by 50% at 200 nM, phytotoxicity can be alleviated by addition of adenine, no inhibitory effect on Escherichia coli | Arabidopsis thaliana | |
additional information | minimal inhibition by inosine monophosphate (IMP) and guanosine monophosphate (GMP) | Arabidopsis thaliana | |
purine nucleotide | allosteric feedback inhibition by end products of the purine biosynthesis pathway, act on the PRPP domain | Arabidopsis thaliana |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.34 | - |
L-glutamine | cell extracts of recombinant AtGPRAT2 expressing Escherichia coli | Arabidopsis thaliana |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
54000 | - |
SDS-PAGE analysis of the recombinant AtGPRAT2 expressed in Escherichia coli | Arabidopsis thaliana |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O | Arabidopsis thaliana | - |
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate | - |
? | |
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O | Arabidopsis thaliana | first step of de novo purine biosynthesis | 5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Arabidopsis thaliana | Q9SI61 | isoform AtGPRAT1 | - |
Arabidopsis thaliana | Q9STG9 | isoform AtGPRAT2, At4g34740 | - |
Arabidopsis thaliana | Q9T0J5 | isoform AtGPRAT3 | - |
Purification (Comment) | Organism |
---|---|
from bacterial extracts by desalting on a Bio-Rad 10DG column in presence of 10 mM dithiothreitol, stored at -70°C | Arabidopsis thaliana |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O | - |
Arabidopsis thaliana | 5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate | - |
? | |
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O | first step of de novo purine biosynthesis | Arabidopsis thaliana | 5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate | - |
? | |
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O | 40 mM glutamine, 5 mM PRPP, 10 mM dithiothreitol | Arabidopsis thaliana | 5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate | glutamate dehydrogenase-coupled assay, glutamate-dependent reduction of 1.36 mM 3-acetylpyridine dinucleotide measurable as increase of absorbance at 363 nm | ? | |
additional information | no GPRAT activity detectable in cell extracts of recombinant AtGPRAT1 expressing Escherichia coli | Arabidopsis thaliana | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
AtGPRT | - |
Arabidopsis thaliana |
Gln phosphoribosylpyrophosphate amidotransferase | - |
Arabidopsis thaliana |
glutamine phosphoribosylpyrophosphate amidotransferase | - |
Arabidopsis thaliana |
GPRAT | - |
Arabidopsis thaliana |
IC50 Value | IC50 Value Maximum | Comment | Organism | Inhibitor | Structure |
---|---|---|---|---|---|
additional information | - |
>500-fold higher for mutant R264K compared to wild-type | Arabidopsis thaliana | DAS734 | |
additional information | - |
20-35 microM leads to 50% inhibition of Arabidopsis root growth (RI50) at in presence of endogenous AtGPRAT2 harbouring R264K polymorphism | Arabidopsis thaliana | DAS734 | |
additional information | - |
200 nM leads to 50% inhibition of Arabidopsis root growth (RI50) in wild-type AtGPRAT2 expressing seedlings | Arabidopsis thaliana | DAS734 | |
0.0002 | - |
10 min preincubation of recombinant enzyme with inhibitor leads to maximum inhibition, slow but reversible binding | Arabidopsis thaliana | DAS734 | |
0.0002 | - |
cell extracts of recombinant AtGPRAT3 expressing Escherichia coli | Arabidopsis thaliana | DAS734 |