Information on EC 4.3.3.6 - pyridoxal 5'-phosphate synthase (glutamine hydrolysing)

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea

EC NUMBER
COMMENTARY hide
4.3.3.6
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RECOMMENDED NAME
GeneOntology No.
pyridoxal 5'-phosphate synthase (glutamine hydrolysing)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
D-ribose 5-phosphate + D-glyceraldehyde 3-phosphate + L-glutamine = pyridoxal 5'-phosphate + L-glutamate + 3 H2O + phosphate
show the reaction diagram
overall reaction
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-
-
D-ribose 5-phosphate + D-glyceraldehyde 3-phosphate + NH3 = pyridoxal 5'-phosphate + 4 H2O + phosphate
show the reaction diagram
(1b)
-
-
-
L-glutamine + H2O = L-glutamate + NH3
show the reaction diagram
(1a)
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
pyridoxal 5'-phosphate biosynthesis II
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vitamin B6 metabolism
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Vitamin B6 metabolism
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SYSTEMATIC NAME
IUBMB Comments
D-ribose 5-phosphate,D-glyceraldehyde 3-phosphate pyridoxal 5'-phosphate-lyase
The ammonia is provided by the glutaminase subunit and channeled to the active site of the lyase subunit by a 100 A tunnel. The enzyme can also use ribulose 5-phosphate and dihydroxyacetone phosphate. The enzyme complex is found in aerobic bacteria, archaea, fungi and plants.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
gene lmo2101 or pdxS, encoding a subunit of pyridoxal 5'-phosphate synthase
UniProt
Manually annotated by BRENDA team
gene Rv2606c
UniProt
Manually annotated by BRENDA team
gene Rv2606c
UniProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
D-ribose 5-phosphate + D-glyceraldehyde 3-phosphate + L-glutamine
pyridoxal 5'-phosphate + L-glutamate + 3 H2O + phosphate
show the reaction diagram
D-ribose 5-phosphate + D-glyceraldehyde 3-phosphate + NH3
pyridoxal 5'-phosphate + 4 H2O + phosphate
show the reaction diagram
-
two proteins form a complex that functions as a glutamine amidotransferase, with YaaE as the glutaminase domain and YaaD as the acceptor and pyridoxal 5'-phosphate synthesis domain. Both the glutaminase and synthase reactions are dependent on the respective protein partner. The synthase reaction can also utilize an external ammonium source but, in contrast to other glutamine amidotransferases, is dependent on YaaE under certain conditions
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-
?
D-ribose 5-phosphate + glyceraldehyd 3-phosphate + NH4+
pyridoxal 5'-phosphate + 4 H2O + phosphate
show the reaction diagram
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-
-
-
?
D-ribose 5-phosphate + glyceraldehyde 3-phosphate + L-glutamine
pyridoxal 5'-phosphate + L-glutamate + 3 H2O + phosphate
show the reaction diagram
D-ribose 5-phosphate + glyceraldehyde 3-phosphate + NH4+
pyridoxal 5'-phosphate + 4 H2O + phosphate
show the reaction diagram
D-ribulose 5-phosphate + D-glyceraldehyde 3-phosphate + L-glutamine
?
show the reaction diagram
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preference of D-ribose 5-phosphate compared to D-ribulose 5-phosphate
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-
?
L-glutamine + H2O
L-glutamate + NH3
show the reaction diagram
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two proteins form a complex that functions as a glutamine amidotransferase, with YaaE as the glutaminase domain and YaaD as the acceptor and pyridoxal 5'-phosphate synthesis domain. Glutaminase activity of YaaE is only detected in the presence of its partner protein YaaD. A 1:1 stoichiometry of both proteins appears to be optimal for activity
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?
ribulose 5-phosphate + glyceraldehyde 3-phosphate + NH3
pyridoxal 5'-phosphate + 4 H2O + phosphate
show the reaction diagram
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-
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?
ribulose 5-phosphate + glyceraldehyde 3-phosphate + NH4+
pyridoxal 5'-phosphate + 4 H2O + phosphate
show the reaction diagram
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
D-ribose 5-phosphate + D-glyceraldehyde 3-phosphate + L-glutamine
pyridoxal 5'-phosphate + L-glutamate + 3 H2O + phosphate
show the reaction diagram
-
the enzyme is involved in vitamin B6 biosynthesis
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?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-deoxy-D-ribose 5-phosphate
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at 12 mM 56% residual activity
4-phospho-D-erythronhydrazide
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acivicin
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inhibition of the glutaminase domain YuaaD, and thus pyridoxal 5'-phosphate synthesis
D-erythronhydrazide
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D-erythrose
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D-erythrose 4-phosphate
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
PdxR
PdxR is a direct activator of the pdxST operon, has a regulatory function in de novo synthesis of pyridoxal 5'-phosphate, encoded by gene pdxR, PdxR is a member of the MocR/GabR subfamily with an N-terminal putative DNA-binding domain and a C-terminal aminotransferase-like domain. PdxR is also a negative autoregulator, and its ability to repress is increased by pyridoxal 5'-phosphate. PdxR-like proteins, for which PLP plays just a signalling role, form a separate functional group among the MocR/GabRtype proteins. Mutational analysis of the PdxR binding site, overview
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.077
D-glyceraldehyde 3-phosphate
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pH 8.0, 37C
0.026 - 0.185
D-ribose 5-phosphate
0.155 - 0.307
glyceraldehyde 3-phosphate
0.93 - 1.54
L-glutamine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0001 - 0.004
D-ribose 5-phosphate
0.0007
D-ribulose 5-phosphate
Bacillus subtilis
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pH 8.0, 37C, free pyridoxal 5'-phosphate synthase subunit (Pdx1)
0.022 - 0.127
L-glutamine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0018 - 0.044
D-ribose 5-phosphate
0.00033 - 0.0039
glyceraldehyde 3-phosphate
0.016 - 0.128
L-glutamine
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.016 - 0.043
4-phospho-D-erythronhydrazide
0.902
D-erythronhydrazide
Plasmodium falciparum
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Pdx1, pH 8.0, 37C
160
D-erythrose
Plasmodium falciparum
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Pdx1, pH 8.0, 37C
3.7
D-erythrose 4-phosphate
Plasmodium falciparum
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Pdx1, pH 8.0, 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00025
0.00031
M103A mutant Pdx1, NH4Cl as NH3+ donor, pyridoxal 5'-phosphate synthesis, pH 8, 37C
0.00042
M103A mutant Pdx1/Pdx2 complex, pyridoxal 5'-phosphate synthesis, pH 8, 37C
0.0007
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Pdx1/Pdx2 complex, pyridoxal 5'-phosphate synthesis, pH 8, 37C
0.000779
wild-type protein, in the presence of Pdx2, pH 8.0, 37C
0.0008
M148L mutant Pdx1, NH4Cl as NH3+ donor, pyridoxal 5'-phosphate synthesis, pH 8, 37C
0.0009
0.0011
Pdx1/Pdx2 complex, pyridoxal 5'-phosphate synthesis, pH 8, 37C
0.0012
L82A mutant Pdx1/Pdx2 complex, pyridoxal 5'-phosphate synthesis, pH 8, 37C
0.0013
0.0015
M46I mutant Pdx1, NH4Cl as NH3+ donor, pyridoxal 5'-phosphate synthesis, pH 8, 37C
0.0018
M19V mutant Pdx1/Pdx2 complex, pyridoxal 5'-phosphate synthesis, pH 8, 37C
0.002
M19V mutant Pdx1, NH4Cl as NH3+ donor, pyridoxal 5'-phosphate synthesis, pH 8, 37C
0.0028
M46I mutant Pdx1/Pdx2 complex, pyridoxal 5'-phosphate synthesis, pH 8, 37C
0.0037
M148L mutant Pdx1/Pdx2 complex, pyridoxal 5'-phosphate synthesis, pH 8, 37C
0.087
M19V mutant Pdx1/Pdx2 complex, glutaminase activity, pH 8, 30C
0.115
L82A mutant Pdx1/Pdx2 complex, glutaminase activity, pH 8, 30C
0.126
M103F mutant Pdx1/Pdx2 complex, glutaminase activity, pH 8, 30C
0.15
Pdx1/Pdx2 complex, glutaminase activity, pH 8, 30C
0.178
M103A mutant Pdx1/Pdx2 complex, glutaminase activity, pH 8, 30C
0.229
M148L mutant Pdx1/Pdx2 complex, glutaminase activity, pH 8, 30C
0.26
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Pdx1/Pdx2 complex, glutaminase activity, pH 8, 30C
0.284
M46I mutant Pdx1/Pdx2 complex, glutaminase activity, pH 8, 30C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Geobacillus kaustophilus (strain HTA426)
Geobacillus kaustophilus (strain HTA426)
Geobacillus kaustophilus (strain HTA426)
Geobacillus kaustophilus (strain HTA426)
Plasmodium berghei (strain Anka)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32830
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unmodified Pdx1, ESI-MS
200000
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dynamic light-scattering analysis
363000
analytical ultracentrifugation, 420000 calculated
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dodecamer
12 * 29000, two interacting hexamers, analytical ultracentrifugation, 35320.7 Da determined by ESI-MS
hexamer
homododecamer
homohexamer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapor diffusion, 3D structure of the pyridoxal 5'-phosphate synthase complex with substrate glutamine bound as well as those of the individual synthase and glutaminase subunits Pdx1 and Pdx2, respectively. The complex is made up of 24 protein units assembled like a cogwheel, a dodecameric Pdx1 to which 12 Pdx2 subunits attach. Macromolecular assembly is directed by an N-terminalalpha-helix on the synthase. Interaction with the synthase subunit leads to glutaminase activation, resulting in formation of an oxyanion hole, a prerequisite for catalysis
P37527 and P37528
purified recombinant enzyme, hanging drop vapour diffusion method, mixing of 00.0015 ml of 22 mg/ml protein in 20 mM Tris-HCl, pH 8.0, and 5 mM 2-mercaptoethanol, with 0.0015 ml of reservoir solution, containing 8% PEG 8000, 0.1 M 3-[cyclohexylamino]-1-propanesulfonic acid, pH 10.5, and 0.2 M sodium chloride, and equilibration against 0.5 ml of reservoir solution, 20C, X-ray diffraction structure determination and analysis at 1.8 A resolution, molecular replacement using the Thermotoga maritima PdxS, PDB code 2ISS
Pdx1, chimeric complex of Pdx1 and Pdx2 from Plasmodium falciparum
chimeric complex of Pdx2 and Pdx1 from Plasmodium berghei
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2-methyl-2,4-pentanediol; crystallized at 23C using 2-methyl-2,4-pentanediol as a precipitant. Crystals of apo and ribose 5'-phosphate complex forms of PdxS diffract to 2.7 A and 3.1 A resolution, respectively, and belong to the monoclinic space group P2(1)
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crystallization at 22C using 2-methyl-2,4-pentanediol as a precipitant. Crystals of PdxS diffract to 2.61 A resolution and belong to the monoclinic space group P2(1), with unit-cell parameters a = 59.30, b = 178.56, c = 109.23 A, beta = 102.97. The asymmetric unit contained six monomers; hanging-drop vapour-diffusion method, 296 K, 2-methyl-2,4-pentanediol
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hanging-drop vapour diffusion method, 16C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity chromatography using the Strep-Tag
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immobilized metal ion affinity chromatography (Ni2+), gel filtration
ion exchange chromatography (Q Sepharose), gel filtration
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recombinant N-terminally His6-tagged enzyme from Escherichia coli strain B834 by nickel affinity chromatography and cleavage of the tag by TEV protease, followed by anion exchange chromatography and gel filtration to about 95% purity
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli Rosetta2 (DE3) pLysS; overexpressed in Escherichia coli strain Rosetta2 (DE3)
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expressed in Escherichia coli Rosetta2 (DE3) pLysS; overexpression in Escherichia coli
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expression in Escherichia coli
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gene btrC2, DNA and amino acid sequence determination, genetic organization, gene btrC2 is not found in the butirosin-biosynthestic gene cluster, sequence comparison, gene complementation of a btrC2 disruptant by Bacillus subtilis gene pdxT, recombinant expression of untagged BtrC2 in Escherichia coli; gene pdxS, DNA and amino acid sequence determination, genetic organization, recombinant expression of untagged protein encoded by pdxS in Escherichia coli
gene lmo2101 or pdxS, encoding a subunit of pyridoxal 5'-phosphate synthase, quantitative PCR expression analysis, determination of transcription start points of genes pdxS and pdxR, overview
gene Rv2606c, recombinant expression of N-terminally His6-tagged enzyme with a TEV protease cleavage site in Escherichia coli strain B834
genes pdxS and pdxT, genetic organization, expression of His-tagged PdxT in Escherichia coli
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His-tagged protein expressed in Escherichia coli BL21 (DE3)
His-tagged protein expressed in Escherichia coli BL21-CodonPlus(DE3)-RIL
His-tagged subunits Pdx1 and Pdx2 expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL
Pdx1 and Pdx2 with and without His-tag expressed
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Strep-Tag-fusion proteins expressed in Escherichia coli BLR (DE3)
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression of pdxS and pdxT is significantly reduced in the presence of pyridoxal or pyridoxal 5'-phosphate, whereas supplementing with pyridoxamine or pyridoxine shows no effect on pdxST expression
the transcription regulator PdxR controls the biosynthesis of pyridoxal or pyridoxal 5'-phosphate in Corynebacterium glutamicum by activating the expression of the pyridoxal or pyridoxal 5'-phosphate synthase genes pdxST
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D99A
mutant of Pdx1 (pyridoxal 5'-phosphate synthase subunit), 5% of the pyridoxal 5'-phosphate synthase activity compared to wild-type enzyme with L-glutamine as N-donor, 17.2% of the glutaminase activity compared to wild-type enzyme
E105D
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Pdx1
E15A
mutant of Pdx2 (glutaminase subunit), 280% of the glutaminase activity compared to wild-type activity, Pdx2 (glutaminase subunit) complex with Pdx1 (pyridoxal 5'-phosphate synthase subunit)
E48A
mutant of Pdx2 (glutaminase subunit), no glutaminase activity in complex with Pdx1 (pyridoxal 5'-phosphate synthase subunit)
H115A
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Pdx1
H115A/R138A
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Pdx1
K149R
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mutant of pyridoxal 5'-phosphate synthase subunit (Pdx1) retains the ability to form the imine adduct
K187A
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Pdx1, 84% of wild-type activity
K18A
mutant of Pdx1 (pyridoxal 5'-phosphate synthase subunit), 40.5% of the pyridoxal 5'-phosphate synthase activity compared to wild-type enzyme with L-glutamine as N-donor, 64.5% of the glutaminase activity compared to wild-type enzyme
K81A
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mutant of pyridoxal 5'-phosphate synthase subunit (Pdx1) does not form the imine adduct
K81R
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mutant of pyridoxal 5'-phosphate synthase subunit (Pdx1) does not form the imine adduct
Q10A
mutant of Pdx2 (glutaminase subunit), 94% of the glutaminase activity compared to wild-type activity, Pdx2 (glutaminase subunit) complex with Pdx1 (pyridoxal 5'-phosphate synthase subunit)
Q10E
mutant of Pdx2 (glutaminase subunit), 2% of the glutaminase activity compared to wild-type activity, Pdx2 (glutaminase subunit) complex with Pdx1 (pyridoxal 5'-phosphate synthase subunit)
Q10N
mutant of Pdx2 (glutaminase subunit), 34% of the glutaminase activity compared to wild-type activity, Pdx2 (glutaminase subunit) complex with Pdx1 (pyridoxal 5'-phosphate synthase subunit)
R106A
mutant of Pdx2 (glutaminase subunit), no glutaminase activity in complex with Pdx1 (pyridoxal 5'-phosphate synthase subunit)
R135A
mutant of Pdx2 (glutaminase subunit), no glutaminase activity in complex with Pdx1 (pyridoxal 5'-phosphate synthase subunit)
R138A
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Pdx1
R288A
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Pdx1, able to activate Pdx2
R288K
-
Pdx1, able to activate Pdx2
S75A
mutant of Pdx1 (pyridoxal 5'-phosphate synthase subunit), 51% of the pyridoxal 5'-phosphate synthase activity compared to wild-type enzyme with L-glutamine as N-donor, 84% of the glutaminase activity compared to wild-type enzyme
D26A/K83A/K151A
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no formation of an internal chromophore with characteristic absorbance at 320 nm during reaction (observed with wild type protein)
DELTA 270-301
mainly monomer, Pdx2 activation, no pyridoxal 5'-phosphate synthesis
DELTA 273-301
dodecamer, Pdx2 activation, no pyridoxal 5'-phosphate synthesis
DELTA 279-301
dodecamer, Pdx2 activation, reduced pyridoxal 5'-phosphate synthesis
DELTA 287-301
dodecamer, Pdx2 activation, precipitates upon addition of glyceraldehyd 3-phosphate
DELTA 293-301
behaviour like DELTA 287-301
DELTA 295-301
behaviour like DELTA 287-301
E136A/R139A/R140A
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formation of an internal chromophore with characteristic absorbance at 320 nm during reaction (also observed with wild type protein)
H196N
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Pdx2, catalytically inactive
R167A
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reduced formation of an internal chromophore with characteristic absorbance at 320 nm during reaction (observed with wild type protein), 50% wild type activity
R85A/H88A/E91A
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no formation of an internal chromophore with characteristic absorbance at 320 nm during reaction (observed with wild type protein), dodecameric assembly prevented
S270A/DELTA 273-301
dodecamer
E116A
activity as the wild-type protein
K117A
no synthesis of pyridoxal 5'-phosphate, capacity to catalyse dihydroxyacetone phosphate isomerization
K148A
no synthesis of pyridoxal 5'-phosphate, capacity to catalyse dihydroxyacetone phosphate isomerization
K240A
activity as the wild-type protein
R136A/R137A
no synthesis of pyridoxal 5'-phosphate, dihydroxyacetone phosphate isomerization activity as the wild-type protein
R164A
completely inactive, dihydroxyacetone phosphate isomerization activity as the wild-type protein
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
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