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Literature summary for 2.4.2.14 extracted from

  • Walsh, T.A.; Bauer, T.; Neal, R.; Merlo, A.O.; Schmitzer, P.R.; Hicks, G.R.; Honma, M.; Matsumura, W.; Wolff, K.; Davies, J.P.
    Chemical genetic identification of glutamine phosphoribosylpyrophosphate amidotransferase as the target for a novel bleaching herbicide in Arabidopsis (2007), Plant Physiol., 144, 1292-1304.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.4.2.14

Cloned(Commentary)

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 (Commentary)

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

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

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 [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 [Da] Molecular Weight Maximum [Da] Comment Organism
54000
-
 SDS-PAGE analysis of the recombinant AtGPRAT2 expressed in Escherichia coli Arabidopsis thaliana

Natural Substrates/ Products (Substrates)

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

Organism UniProt Comment Textmining
Arabidopsis thaliana Q9SI61 isoform AtGPRAT1
-
Arabidopsis thaliana Q9STG9 isoform AtGPRAT2, At4g34740
-
Arabidopsis thaliana Q9T0J5 isoform AtGPRAT3
-

Purification (Commentary)

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 and Products (Substrate)

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

Synonyms Comment Organism
AtGPRT
-
 Arabidopsis thaliana
Gln phosphoribosylpyrophosphate amidotransferase
-
 Arabidopsis thaliana
glutamine phosphoribosylpyrophosphate amidotransferase
-
 Arabidopsis thaliana
GPRAT
-
 Arabidopsis thaliana

IC50 Value

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