Information on EC 4.2.1.75 - uroporphyrinogen-III synthase

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

EC NUMBER
COMMENTARY hide
4.2.1.75
-
RECOMMENDED NAME
GeneOntology No.
uroporphyrinogen-III synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydroxymethylbilane = uroporphyrinogen III + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cyclization
-
-
elimination
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
-
heme metabolism
-
-
Metabolic pathways
-
-
Porphyrin and chlorophyll metabolism
-
-
tetrapyrrole biosynthesis I (from glutamate)
-
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tetrapyrrole biosynthesis II (from glycine)
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SYSTEMATIC NAME
IUBMB Comments
hydroxymethylbilane hydro-lyase (cyclizing; uroporphyrinogen-III-forming)
In the presence of EC 2.5.1.61, hydroxymethylbilane synthase, the enzyme forms uroporphyrinogen III from porphobilinogen.
CAS REGISTRY NUMBER
COMMENTARY hide
37340-55-9
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
uroporphyrinogen III synthase/methyltransferase gene cobA/hemD
UniProt
Manually annotated by BRENDA team
uroporphyrinogen III synthase/methyltransferase gene cobA/hemD
UniProt
Manually annotated by BRENDA team
pv. tomato, gene pspto_0129
-
-
Manually annotated by BRENDA team
pv. tomato, gene pspto_0129
-
-
Manually annotated by BRENDA team
wheat
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Hydroxymethylbilane
?
show the reaction diagram
Hydroxymethylbilane
Uroporphyrinogen III
show the reaction diagram
hydroxymethylbilane
uroporphyrinogen III + H2O
show the reaction diagram
hydroxymethylbilane
uroporphyrinogen-III + H2O
show the reaction diagram
Preuroporphyrinogen
Uroporphyrinogen III
show the reaction diagram
additional information
?
-
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
Hydroxymethylbilane
?
show the reaction diagram
Hydroxymethylbilane
Uroporphyrinogen III
show the reaction diagram
Q72KM1
-
-
-
?
hydroxymethylbilane
uroporphyrinogen III + H2O
show the reaction diagram
hydroxymethylbilane
uroporphyrinogen-III + H2O
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K+
-
activates
KCl
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optimal concentration is 100 mM, wild-type enzyme
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,5'-dithiobis(2-nitrobenzoate)
-
-
Butane-2,3-dione
Coproporphyrins
-
-
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diethyl dicarbonate
hydroxylamine
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iodoacetamide
-
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iodoacetic acid
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Macrocyclic spiro-lactam derivatives
-
-
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N-ethylmaleimide
NH4+
-
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p-chloromercuribenzoate
p-hydroxymercuribenzoate
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Phenylglyoxal
pyridoxal 5'-phosphate
sulphydryl reagents
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uroporphyrinogen III
Uroporphyrins
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-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00015 - 10
Hydroxymethylbilane
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.6 - 1200
Hydroxymethylbilane
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1 - 16000
Hydroxymethylbilane
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.25
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purified recombinant mutant Y166F
9.4
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hydroxymethylbilane
25
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hydroxymethylbilane
33.37
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pH not specified in the publication, temperature not specified in the publication
40
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purified recombinant wild-type
54.7
-
-
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.7 - 7.9
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-
7.8 - 7.95
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-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9.5
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6.5 - 8.5
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at pH 6.5: 10% of optimum activity, at pH 8.5: 30% of optimum activity
7 - 9
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at pH 7.0: 60% of optimum activity, at pH 9.0: 60% of optimum activity
7.5 - 9
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pH 7.5: about 60% of maximal activity, pH 9.0: about 70% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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enzyme assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37 - 52
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the isothermal unfolding rate is determined in the range of 37C-52C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
uroporphyrinogen III synthase forms pairs of bilateral stripes in the lateral mesoderm at the 15-somite stage. At 24 h post-fertilization, uroporphyrinogen III synthase is predominantly expressed in the intermediate cell mass (ICM) that is the major site of hematopoiesis. The expression is drastically suppressed in the bloodless mutants cloche and vlad tepes/gata 1 from 15-somite to 24hpf stages, indicating that both cloche abd vlad tepes/gata 1 are required for the induction and maitenance of uroporphyrinogen III synthase expression in intermediate cell mass
Manually annotated by BRENDA team
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erythroleukemia cells
Manually annotated by BRENDA team
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erythroleukemia cells
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
exclusive localization in chloroplasts, UROS has an N-terminal extension acting as a targeting sequence for import of the precursor protein of 34 kD into chloroplasts, followed by processing to the mature size of 29 kDa
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas syringae pv. tomato (strain DC3000)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
26000
-
SDS-PAGE
27890
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nucleotide sequence
28810
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mass spectrometry
29000
-
gel filtration
29100
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nucleotide sequence
29500
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SDS-PAGE
30000
-
gel filtration
31000
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SDS-PAGE
32000
-
SDS-PAGE
36000
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gel filtration
38500
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SDS-PAGE, gel filtration
41500
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gel filtration
42000
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gel filtration
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
the N-terminal extension of UROS is processed resulting in the mature enzyme of 29 kDa
additional information
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UROIIIS is processed via the proteosome pathway, which can be reverted by reversibly inhibiting the proteosome with the aldehyde MG132
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
5 mg/ml purified recombinant wild-type and selenomethionine-enzyme, sitting drop vapour diffusion method, 4C, equal volumes of protein solution, containing 10 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, and reservoir solution, containing 20% 2-methyl-2,4-pentanediol, 100 mM MES, pH 6.0, X-ray diffraction structure determination and analysis at 1.85-2.1 A resolution
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purified wild-type recombinant His6-tagged U3S and selenomethionine-substituted derivative, 0.001 ml of 20 mg/ml protein in 20 mM Hepes, pH 7.2, 20 mM NaCl, is mixed with 0.001 ml of reservoir solution containing 20% PEG 3350, 0.2 M tri-Na citrate, and equilibrated against 0.1 ml of reservoir solution, about a month at 8C and followed by microseeding method, X-ray diffraction structure determination and analysis at 2.5 A resolution, single wavelength anomalous dispersion method
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purified recombinant enzyme, three apoenzyme forms, U3S1, U3S2, U3S3, and one enzyme-product complex form, vapor diffusion in sitting drop method, mixing 0.002 ml of protein with 0.002 ml of precipitant solution and equilibration over 1 mL of precipitant solution of different compositions, for U3S1, 1.2 M NaH2PO4, 0.8 M K2H2PO4, for U3S2, 21-24% PEG 8000, 0.2 M Mg-acetate, 0.1 M MES-HCl, pH 6.5, 0.2 M NaCl, and for U3S3, 18-24% PEG 8000, 0.1 M Tris-HCl, pH 8.5, 0.2 mM MgCl2, 4C, 1-7 days, X-ray diffraction structure determination and analysis at 1.6-2.0 A resolution
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
-
20% loss of activity in 10 min
35
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enzyme reaction linear up to 30 min
37
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about 20% loss of activity after 48 h, about 50% loss of activity after 72 h, buffer containing 1 mM DTT
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
10% glycerol stabilizes the purified recombinant enzyme in 20 mM sodium phosphate, pH 7.45, 100 mM NaCl, 2 mM DTT, and 0.1 mM EDTA at -80C
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a helical region in the molecule is essential to retain the kinetic stability of the folded conformation
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activity does not change after several cycles of freeze-and thawing in potassium phosphate buffer, pH 7.4, containing DTT and glycerol
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dithiothreitol stabilizes
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enzyme reaction linear up to 30 min at 35C
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human erythrocyte enzyme more stable than wheat germ enzyme
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purification needs to be carried out as close to 0C as possible
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recombinant enzyme in cell lysate is unstable, glycerol stabilizes
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
not affected by N-bromosuccinimide oxidation
-
5883
photooxidation in presence of Rose Bengal
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, several months
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-20C, stable to freezing
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-70C, at least two years
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-70C, several weeks
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4 C, stable for one week without significant change of activity
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80C, stable for at least one year
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; recombinant N-terminally His-tagged enzyme from Escherichia coli strain strain BL21(DE3) by ultrafiltration, nickel affinity and anion exchange chromatography, followed by gel filtration to homogeneity
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expression in Escherichia coli
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from overproducing strain
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Ni-NTA column chromatography and Superdex 75 gel filtration
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partial
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recombinant
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recombinant His-tagged enzyme from Escherichia coli
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity and anion exchange chromatography, and gel filtration to homogeneity
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, the tag is cleaved off by TEV, followed by gel filtration
recombinant His6-tagged enzyme from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography, anion exchange chromatography, and gel filtration
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recombinant wild-type UROIIIS and 25 point mutants in Escherichia coli to over 97% purity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, expression of wild-type and mutant enzymes
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expressed in Escherichia coli C41(DE3) cells
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expression in Escherichia coli
expression in Escherichia coli; expression of N-terminally His-tagged enzyme in Escherichia coli strain strain BL21(DE3)
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expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
expression of His-tagged wild-type and mutant enzymes in Escherichia coli
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expression of the N-terminal His-tagged wild-type and mutant enzymes in Escherichia coli BL21(DE3)
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expression of wild-type UROIIIS and 25 point mutants in Escherichia coli
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gene cobA/hemD encoding the bifunctional uroporphyrinogen III synthase/methyltransferase, DNA and amino acid sequence determination and analysis, subcloning in Escherichia coli strain DH5alpha, expression in Escherichia coli strain BL21(DE3). Unsuccessful expressionin Lactococcus lactis strain NZ9000
gene HEM4, library screening and complementation studies, sequence comparisons, expression of the enzyme in the knockout Saccharomyces cerevisiae S150-2B strain, overvexpression of the His-tagged enzyme in Escherichia coli BL21(DE3) cells
gene pspto_0129, expression of His6-tagged enzyme in Escherichia coli strain BL21 (DE3). The selenomethionine-substituted derivative is produced by expression in an Escherichia coli methionine auxotrophic strain B834(DE3)
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overexpression as N-terminally his-tagged enzyme in Escherichia coli
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overproduction
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semi-quantitative real-time PCR analysis of ubiquitous and erythroid-specific UROS mRNAs, expression analysis
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stably expression of GFP-tagged UROIIIS-WT or UROIIIS-C73R proteins in MLP29cell lines, reduced expression levels of C73R-UROIIIS in the mammalian cells
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
branchpoint sequence mutation markedly reduces the wild type transcript and enzyme activity
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the efficiency of Uros gene suppression is developmentally regulated, being most effective in mature seedling leaves compared with newly emergent leaves
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the hepatic and splenic URO-synthase activities are normal or elevated (1-1.6fold) compared with age and sex-matched feline control activities in the dominantly inherited acute intermittent porphyria lines
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C73D
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computational modeling, structure comparison with the wild-type enzyme and the other C73 mutants
C73L
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computational modeling, structure comparison with the wild-type enzyme and the other C73 mutants
C73N
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computational modeling, structure comparison with the wild-type enzyme and the other C73 mutants
C73R/A69E
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the mutant shows reduced activity compared to the wild type enzyme
C73R/L43D
-
the mutant shows slightly increased activity compared to the wild type enzyme
C73Y
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computational modeling, structure comparison with the wild-type enzyme and the other C73 mutants
E127A
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site-directed mutagenesis, unaltered activity level compared to the wild-type enzyme
E249stop
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clinical mutation, truncated protein, loss of helix 12
K220A
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site-directed mutagenesis, slightly increased activity compared to the wild-type enzyme
Q249X
-
the mutation is associated with congenital erythropoietic porphyria
R65A
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site-directed mutagenesis, slightly reduced activity compared to the wild-type enzyme
S197A
-
site-directed mutagenesis, unaltered activity level compared to the wild-type enzyme
S63A
-
site-directed mutagenesis, unaltered activity level compared to the wild-type enzyme
T103A
-
site-directed mutagenesis, reduced activity compared to the wild-type enzyme
T227A
-
site-directed mutagenesis, slightly increased activity compared to the wild-type enzyme
T228A
-
site-directed mutagenesis, conserved residue near to the active site cleft, reduced activity compared to the wild-type enzyme
Y168A
-
site-directed mutagenesis, reduced activity compared to the wild-type enzyme
C73R
0.24% of wild-type activity, homozygous mice are fetal lethals
V99A
5.9% of wild-type activity, homozygous mice are fetal lethals
V99L
14.8% of wild-type activity
E114A
-
site-directed mutagenesis, the mutant shows activity slightly reduced compared to the wild-type enzyme
E38A
-
site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme
L116A
-
site-directed mutagenesis, the mutant shows activity slightly reduced compared to the wild-type enzyme
R164A
-
site-directed mutagenesis, the mutation causes a decrease in enzymatic activity to about 70% that of the wild-type enzyme
R219A
-
site-directed mutagenesis, the mutation causes a decrease in enzymatic activity to about 25% that of the wild-type enzyme
E114A
-
site-directed mutagenesis, the mutant shows activity slightly reduced compared to the wild-type enzyme
-
E38A
-
site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme
-
L116A
-
site-directed mutagenesis, the mutant shows activity slightly reduced compared to the wild-type enzyme
-
R164A
-
site-directed mutagenesis, the mutation causes a decrease in enzymatic activity to about 70% that of the wild-type enzyme
-
R219A
-
site-directed mutagenesis, the mutation causes a decrease in enzymatic activity to about 25% that of the wild-type enzyme
-
D17A
-
60.3% of wild-type activity
E68A
-
82.8% of wild-type activity
H173L
-
106.9% of wild-type activity
K10Q
-
39.7% of wild-type activity
K147Q
-
34.5% of wild-type activity
N121D
-
93.1% of wild-type activity
Y168F
-
1.7% of wild-type activity
Y97F
-
74.1% of wild-type activity
D112G
-
site-directed mutagenesis, 50% reduced activity compared to the wild-type enzyme
R15Q
-
site-directed mutagenesis, unaltered activity level compared to the wild-type enzyme
S68A
-
site-directed mutagenesis, unaltered activity level compared to the wild-type enzyme
T100A
-
site-directed mutagenesis, 75% reduced activity compared to the wild-type enzyme
Y166F
-
site-directed mutagenesis, more than 99% reduced activity compared to the wild-type enzyme
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged wild-type enzyme from Escherichia coli, to homogeneity
-
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