Information on EC 1.2.1.70 - glutamyl-tRNA reductase

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

EC NUMBER
COMMENTARY hide
1.2.1.70
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RECOMMENDED NAME
GeneOntology No.
glutamyl-tRNA reductase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-glutamate 1-semialdehyde + NADP+ + tRNAGlu = L-glutamyl-tRNAGlu + NADPH + H+
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
tetrapyrrole biosynthesis I (from glutamate)
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heme metabolism
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Porphyrin and chlorophyll metabolism
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Metabolic pathways
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Biosynthesis of secondary metabolites
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
L-glutamate-semialdehyde:NADP+ oxidoreductase (L-glutamyl-tRNAGlu-forming)
This enzyme forms part of the pathway for the biosynthesis of 5-aminolevulinate from glutamate, known as the C5 pathway. The route shown in the diagram is used in most eubacteria, and in all archaebacteria, algae and plants. However, in the alpha-proteobacteria, EC 2.3.1.37, 5-aminolevulinate synthase, is used in an alternative route to produce the product 5-aminolevulinate from succinyl-CoA and glycine. This route is found in the mitochondria of fungi and animals, organelles that are considered to be derived from an endosymbiotic alpha-proteobacterium. Although higher plants do not possess EC 2.3.1.37, the protistan Euglena gracilis possesses both the C5 pathway and EC 2.3.1.37.
CAS REGISTRY NUMBER
COMMENTARY hide
119940-26-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
subspecies asukaensis
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Manually annotated by BRENDA team
PCC 6803
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
additional information
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three mechanisms for plant GluTR activity regulation: (i) the end-product feedback inhibition by heme, (ii) repression by a membrane protein FLUORESCENT (FLU), and (iii) formation of complex with a soluble GluTR-binding protein (GBP)
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
L-glutamate 1-semialdehyde + NADP+ + tRNAGlu
L-glutamyl-tRNAGlu + NADPH + H+
show the reaction diagram
L-glutaminyl-tRNAGlu + NADPH + H+
? + NADP+ + tRNAGlu
show the reaction diagram
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?
L-glutamyl-tRNAGlu + NADH + H+
L-glutamate 1-semialdehyde + NAD+ + tRNAGlu
show the reaction diagram
L-glutamyl-tRNAGlu + NADPH + H+
L-glutamate 1-semialdehyde + NADP+ + tRNAGlu
show the reaction diagram
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
L-glutamate 1-semialdehyde + NADP+ + tRNAGlu
L-glutamyl-tRNAGlu + NADPH + H+
show the reaction diagram
L-glutamyl-tRNAGlu + NADPH + H+
L-glutamate 1-semialdehyde + NADP+ + tRNAGlu
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADP+
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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restores activity after treatment with chelating agents
Mn2+
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restores activity after treatment with chelating agents
additional information
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no significant stimulation by high salt concentrations
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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5 mM, 25% inhibition
2,2'-dipyridyl
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5 mM, 20% inhibition
2,4-diphenyl-6-styryl-1-p-tolyl-pyridinium boron tetrafluoride
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IC50: 0.01 mM
4-[4-(3,4-dihydroxyphenyl)-2,3-dimethylbutyl]benzene-1,2-diol
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IC50: 0.055 mM
5,5'-dithiobis(2-nitrobenzoic acid)
5,5'-dithiobis-(2-nitrobenzoic acid)
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5,6-dichloro-1,3-benzodioxol-2-one
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IC50: 0.055 mM
Cd2+
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1 mM Cd(Ac)2, 92% inhibition
Cu2+
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1 mM CuCl2, 84% inhibition
EDTA
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10 mM, 55% inhibition
EGTA
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10 mM, 35% inhibition
Fe2+
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5 mM FeSO4, 66% inhibition
Fe3+
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10 mM FeCl3, 80% inhibition
glutamate 1-semialdehyde
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0.2 mM, 50% inhibition
glutamate-1-semialdehyde
1.0 mM, 50% inhibition
glutamycin
H2O2
inactivates the enzyme. H2O2 decreases the stimulation of GluTR by glutamate semialdehyde 1-2 aminomutase, GSAM
Hemin
50% inhibition at 0.0015 mM
iodoacetamide
N-tosyl-L-phenylalaninechloromethyl ketone
0.1 mM, 90% inhibition, 1.0 mM, complete inhibition
PbCl2
0.1 mM, 60% inhibition, 1.0 mM, complete inhibition
protein FLU
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protein GBP
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a soluble GluTR-binding protein, enzyme binding structure, overview. the GluTR-GBP complex is stable and has a low apparent dissociation constant. Protein GBP is initially found in chloroplast stroma
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PtCl4
Zn2+
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2.5 mM, ZnSO4, 94% inhibition
ZnCl2
0.2 mM, 45% inhibition, 5.0 mM, 90% inhibition
additional information
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structure analysis of the FLUTPR-GluTR-GBP ternary complex, overview. Three mechanisms for plant GluTR activity regulation: (i) the end-product feedback inhibition by heme, (ii) repression by a membrane protein FLUORESCENT (FLU), and (iii) formation of complex with a soluble GluTR-binding protein (GBP)
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cycloheximide
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GluBP regulator
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the GluTR regulator, GluTR binding protein (GluBP), spatially organizes tetrapyrrole synthesis by distributing enzyme GluTR into different suborganellar locations. GluBP belongs to a heme-binding family involved in heme metabolism. Complex structure of GluTR-GluBP from Arabidopsis thaliana, overview. The dimeric GluBP binds symmetrically to the catalytic domains of the V-shaped GluTR dimer via its C-terminal domain. A substantial conformational change of the GluTR NADPH-binding domain is observed, confirming the postulated rotation of the NADPH-binding domain for hydride transfer from NADPH to the substrate. Arg146, guarding the door for metabolic channeling, adopts alternative conformations, which may represent steps involved in substrate recognition and product release. GluBP stimulates GluTR catalytic efficiency with an approximate 3fold increase of the 5-aminolevulinic acid formation rate. Tunnel formation in the GluTR-GluBP complex for release of product L-glutamate 1-semialdehyde. GluBP stimulates GluTR activity and regulates GSA release
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glutamate-1-semialdehyde aminotransferase
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heme
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under high heme requirement for respiration levels of GluTR increase
Ozone
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induces expression in photosynthetic tissues
protein GSAM
stimulation of GluTR by glutamate semialdehyde 1-2 aminomutase, GSAM. GluTR activity is stimulated upon formation ofa complex with protein GSAM. This effect is observed only when GluTR contained a heme/protein ratio of 1/12. GluTR activity and the stimulation of this enzyme by protein GSAM are reduced by treatment with H2O2
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additional information
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is induced in photosynthetic tissues by oxidative stresses such as wounding
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.024
L-glutamyl-tRNAGlu
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pH 8.1, 37°C
0.039
NADPH
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pH 8.1, 37°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.13
L-glutamyl-tRNAGlu
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pH 8.1, 37°C
0.15
NADPH
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pH 8.1, 37°C
additional information
additional information
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01
2,4-diphenyl-6-styryl-1-p-tolyl-pyridinium boron tetrafluoride
Arabidopsis thaliana
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IC50: 0.01 mM
0.055
4-[4-(3,4-dihydroxyphenyl)-2,3-dimethylbutyl]benzene-1,2-diol
Arabidopsis thaliana
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IC50: 0.055 mM
0.055
5,6-dichloro-1,3-benzodioxol-2-one
Arabidopsis thaliana
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IC50: 0.055 mM
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.12
fusion protein with glutathione S-transferase
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
isoelectric focusing
8.55
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sequence calculation
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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kinetin but not cytokinin stimulates expression of glutamyl-tRNA reductase expression in etiolated plants
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
39000
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x * 39000, SDS-PAGE
45436
x * 45436, electrospray ionization mass spectrometry
46000
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2 * 52500 + 2 * 46000, sucrose gradient sedimentation of glutamyl-tRNA reductase preincubated with glutamate-1-semialdehyde aminotransferase. Both enzyme also co-precipitate in immunoprecipitation experiments
48000
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2 * 48000, homodimer with an extended structure, SDS-PAGE
48448
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2 * 48448, homodimer with an extended structure, electrospray ionization mass spectrometry
49000
SDS-PAGE, recombinant protein
50000
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SDS–PAGE of His6-tagged GluTR
50490
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MALDI-TOF mass spectrometry of His6-tagged GluTR
54000
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x * 54000, SDS-PAGE
60000
4 * 60000, SDS-PAGE
101000
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sucrose density gradient sedimentation, dimer
104000
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cross-linking of GluTR with glutaraldehyde
130000
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glycerol gradient sedimentation, gel filtration
148000
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relative molecular mass of native recombinant GluTR after removal of His6-tag, determined by gel permeation chromatography
180000
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gel filtration
190000
gel filtration
250000
gel filtration
270000
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gel filtration
350000
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glycerol density gradient centrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotetramer
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2 * 52500 + 2 * 46000, sucrose gradient sedimentation of glutamyl-tRNA reductase preincubated with glutamate-1-semialdehyde aminotransferase. Both enzyme also co-precipitate in immunoprecipitation experiments
homodimer
monomer
tetramer
4 * 60000, SDS-PAGE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified GluTR in complex with GluBP, X-ray diffraction structure determination and analysis at 2.8 A resolution, modeling
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purified recombinant GluTR in ternary complex with GBP and FLUTPR, the protein are mixed at molar ratio of 2:3:3, X-ray diffraction structure determination and analysis at 3.2 A resolution, molecular replacement method
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uncomplexed TPR domain of FLU (FLUTPR) and complex of the dimeric domain of GluTR bound to FLUTPR, hanging drop vapor diffusion method, from 0.2 M NaCl, 0.1 M Bis-Tris, pH 6.5, and 25% w/v PEG 3350 in 1 week, and from 0.15 M KBr and 30% w/v PEG monomethyl ether 2000, in 3 weeks, X-ray diffraction structure determination and analysis at 1.45 and 2.4 A resolution, respectively, molecular replacement and modeling
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crystallized in presence of glutamycin, the structure is solved by the multiple isomorphous replacement method using three heavy atom derivatives. The structure is subsequently refined at a resolution of 1.95 A
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, 6*His-tagged enzyme is stable for at least 6 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by Ni2+-Sepharose affinity chromatography to near homogeneity
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fusion protein with glutathione S-transferase
Ni-affinity chromatography
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant N-terminally His6-tagged FLUTPR and GluTRDD in Escherichia coli strain BL21(DE3)
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wild-type GluTR and variants
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
AtHemA1 overexpression in Nicotiana tabacum cv Samsun NN using Agrobacterium tumefaciens and Arabidopsis thaliana by transformation
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DNA and amino acid sequence determination and analysis, phylogenetic tree
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expression as His-tag fusion protein in Escherichia coli
expression in Escherichia coli
expression in Escherichia coli as a fusion protein with glutathione S-transferase
expression in Escherichia coli BL21
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expression plasmid pBKCwt overexpression of a 6*His-tagged enzyme
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gene HEMA1, recombinant co-overexpression of N-terminally His6-tagged FLUTPR and GluTRDD in Escherichia coli strain BL21(DE3)
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hemA ligated into pUC19 vector with a Lys insert between Thr-2 and Leu-3 at N terminus, overexpression in Escherichia coli
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His(6)-GluTR overexpressed in Escherichia coli BL21 (DE3)
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N-terminal His6-tagged GluTR expressed in Escherichia coli
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overexpression in Escherichia coli
recombinant enzyme expression in Rhodobacter sphaeroides strain DSM 5864, which synthesizes 5-aminolevulinic acid through the C-4 pathway and also has the genes coding for the enzymes of the C-5 pathway, except for glutamyl-tRNA reductase. Expression of the glutamyl-tRNA reductase gene from Rhodospirillum rubrum enabled the C-5 pathway in Rhodobacter sphaeroides to function upon assembling all the required genes
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recombinant overexpression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)
wild-type GluTR and variants produced as N-terminal His6-fusion proteins
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
etiolation of initially green dark-grown larch cotyledons is connected with decreasing content of glutamyl-tRNA reductase
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expression of HEMA1 that encodes the dominant GluTR in the photosynthetic tissues is regulated by light
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expression profiles in the first hours of deetiolation of Arabidopsis seedlings show an abundance of GluTR that gradually increased with chlorophyll biosynthesis
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the relative amount of GluTR is similar both in the dark-grown and light/dark-grown gabaculine-treated seedlings
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C48A
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no activity
C170S
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mutant enzyme with esterase activity 95% of the wild-type activity and reductase activity with 90% of the wild-type activity
C74S
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mutant enzyme with esterase activity 110% of the wild-type activity and reductase activity with 120% of the wild-type activity
E114K
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mutant enzyme with no esterase and reductase activity
E54K
retains 6% reductase and 2% esterase activity
G106N
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mutant enzyme with no esterase and reductase activity
G191D
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mutant enzyme reveals esterase, 105% of the wild-type activity, but no reductase activity
G44C/S105N/A326T
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mutant enzyme with no esterase and reductase activity
G7D
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mutant enzyme with no esterase and reductase activity
H99N
retains 5% reductase and 4% esterase activity
Q116L
lacks reductase activity whereas 30% esterase activity is retained
R314C
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mutant enzyme with no esterase and reductase activity
R52K
retains 5% reductase and 4% esterase activity
S109A
retains 28% reductase and 30% esterase activity
S145F
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mutant enzyme with no esterase and reductase activity
S22L/S164F
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mutant enzyme with no esterase and reductase activity
T49V
retains 10% reductase and 5% esterase activity
I318L/R322G/N454D
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mutant enzyme with greatly reduced activity
I464P
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mutant enzyme with greatly reduced activity
L387H/L302S
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mutant enzyme with greatly reduced activity
M122K/K154N/F371L/E400K
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mutant enzyme with greatly reduced activity
C393S
95% of the GluTR reductase activity compared to wild-type enzyme, 100% of the GluTR esterase activity compared to wild-type enzyme
C42S
no GluTR reductase and GluTR esterase activity
C6S
130% of the GluTR reductase activity compared to wild-type enzyme, 120% of the GluTR esterase activity compared to wild-type enzyme
C90S
85% of the GluTR reductase activity compared to wild-type enzyme, 105% of the GluTR esterase activity compared to wild-type enzyme
H84A
no GluTR reductase activity, 5% of the GluTR esterase activity compared to wild-type enzyme
H84N
30% of the GluTR reductase activity compared to wild-type enzyme, 15% of the GluTR esterase activity compared to wild-type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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recombinant Escherichia coli allows efficient production of 5-aminolevulinic acid directly from glucose
synthesis
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the recombinant engineered GluTR variant R1 can be used for improvement of the C5 pathway to enhance 5-aminolevulinic acid and other products
additional information
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