Information on EC 1.3.1.91 - tRNA-dihydrouridine20 synthase [NAD(P)+]

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

EC NUMBER
COMMENTARY hide
1.3.1.91
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RECOMMENDED NAME
GeneOntology No.
tRNA-dihydrouridine20 synthase [NAD(P)+]
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
5,6-dihydrouracil20 in tRNA + NAD(P)+ = uracil20 in tRNA + NAD(P)H + H+
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
tRNA-5,6-dihydrouracil20:NAD(P)+ oxidoreductase
A flavoenzyme [3]. The enzyme specifically modifies uracil20 in tRNA.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
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
uracil in pre-tRNALeu + NAD(P)H + H+
5,6-dihydrouracil in pre-tRNALeu + NAD(P)+
show the reaction diagram
-
-
-
-
?
uracil in pre-tRNATyr + NAD(P)H + H+
5,6-dihydrouracil in pre-tRNATyr + NAD(P)+
show the reaction diagram
-
-
-
-
?
uracil in tRNA + NAD(P)H + H+
5,6-dihydrouracil in tRNA + NAD(P)+
show the reaction diagram
uracil20 in tRNA + NAD(P)H + H+
5,6-dihydrouracil20 in tRNA + NAD(P)+
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
uracil in tRNA + NAD(P)H + H+
5,6-dihydrouracil in tRNA + NAD(P)+
show the reaction diagram
-
-
-
-
?
uracil20 in tRNA + NAD(P)H + H+
5,6-dihydrouracil20 in tRNA + NAD(P)+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FMN
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the flavin cofactor is involved in the reduction of uridine, FMN binding site structure, overview. HsDus2dusD binds the FMN cofactor non-covalently at the C-terminal end of the beta-barrel, above beta-strands 1 and 8
NAD(P)H
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
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Michaelis-Menten kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
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kinetics of reductive and oxidative half-reactions
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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isozyme Dus2 is upregulated
Manually annotated by BRENDA team
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overexpression of hDUS2 in non–small cell lung carcinomas is identified by analysis of the gene-expression profiles
Manually annotated by BRENDA team
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elevated hDus2 mRNA and protein levels
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
43000
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x * 43000, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant His-tagged and SeMet-labeled DusC, sitting drop vapour diffusion method, mixing of 200 nl of about 10 mg/ml of protein in 20 mM HEPES, pH 7.6, 1 mM MgCl2, 200 mM KCl, 7 mM 2-mercaptoethanol, and 10% glycerol, with 200 nl of reservoir solution containing 0.1 M Tris, pH 7.9, 0.2 M sodium acetate, and 12% PEG 4000 for the wild-type enzyme and 0.1 M imidazole, pH 8.0, 15% v/v 2-propanol, and 20% v/v glycerol for the SeMet-labeled enzyme, X-ray diffraction structure determination and analysis at 2.1 A resolution
purified recombinant catalytic HsDus2dusD domain and tRNA binding domain HsDus2dsRBD (Glu347-Lys451), by hanging drop vapor diffusion method, at 19°C, X-ray diffraction structure determination and crystal structure analysis, PDB IDs 4WFS and 4WFT
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purified recombinant hDus2 catalytic and tRNA-recognition domains (residues 1-340), sitting drop vapour diffusion method, 300 nl of 10 mg/ml protein in 20 mM Tris, pH 8.0, 100 mM NaCl, 5 mM imidazole, and 5 mM DTT, are mixed with 0. 54 ml of reservoir solution containing 0.1 M MES-malic acid-Tris, pH 4.0, and 25% w/v PEG 1500, 2 days, 19°C, X-ray diffraction structure determination and analysis by single-wavelength anomalous diffraction at 1.9 A resolution, automated molecular replacement with different search models, and modeling
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme
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recombinant His-tagged enzyme by nickel affinity chromatography and gel filtration
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recombinant His-tagged wild-type and selenomethionine-labeled enzymes from Escherichia coli cell-free extract (centrifugation at 40000 x g) by nickel affinity chromatography, dialysis, heparin affinity chromatography, and gel filtration
recombinant wild-type and selenomethionine-labeled hDus2 1-340 fragment, comprising the hDus2 catalytic and tRNA-recognition domains, from Escherichia coli strains BL21(DE3) and B834(DE3), respectively
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression in Escherichia coli. There is a higher proportion of tRNAGly in Escherichia coli expressinf DUS2
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recombinant expression of His-tagged enzyme as wild-type and selenomethionine-labeled proteins in Escherichia coli strains BL21(DE3) and B834 (DE3), respectively
recombinant expression of His-tagged enzyme, functional complementation of an enzyme-deficient Saccharomyces cerevisiae mutant by expression of enzyme domains HsDus2dusD and HsDus2dsRBD, both domains are required for activity
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recombinant expression of wild-type and selenomethionine-labeled hDus2 1-340 fragment, comprising the hDus2 catalytic and tRNA-recognition domains, in Escherichia coli strains BL21(DE3) and B834(DE3), respectively
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
upregulation of hDUS2 is a relatively common feature of pulmonary carcinogenesis
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C117A
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rate constant is 1600fold lower than the rate constant of the wild-type enzyme
additional information
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siRNA-dependent knockdown of hDus2 decreases colony formation and cell viability of non-small cell lung cancer cells, while hDus2 immunohistochemical staining correlated with patient survival
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
diagnostics
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the enzyme defined as an independent prognostic factor for the development of non-small cell lung cancer cells
medicine
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