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Enzyme Nomenclature
Information on EC 1.3.1.91 - tRNA-dihydrouridine20 synthase [NAD(P)+]
for references in articles please use BRENDA:EC1.3.1.91
Word Map on EC 1.3.1.91
- 1.3.1.91
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d-loops
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thermophilus
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thermus
- trnaphe
- flavin
- archaea
- carcinogenesis
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l-shaped
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unmodified
- medicine
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double-stranded
- diagnostics
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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
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EC NUMBER 
COMMENTARY 
1.3.1.91
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RECOMMENDED NAME
GeneOntology No.
tRNA-dihydrouridine20 synthase [NAD(P)+]
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
5,6-dihydrouracil20 in tRNA + NAD(P)+ = uracil20 in tRNA + NAD(P)H + H+
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5,6-dihydrouracil20 in tRNA + NAD(P)+ = uracil20 in tRNA + NAD(P)H + H+
Dus proteins adopt a common substrate recognition mechanism using an adapter molecule, whereas the manner of tRNA binding is diverse
5,6-dihydrouracil20 in tRNA + NAD(P)+ = uracil20 in tRNA + NAD(P)H + H+
dsRNA binding mode, modeling
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SYSTEMATIC NAME
IUBMB Comments
tRNA-5,6-dihydrouracil20:NAD(P)+ oxidoreductase
A flavoenzyme [3]. The enzyme specifically modifies uracil20 in tRNA.
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
physiological function
additional information
evolution
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In humans, there are four Dus enzymes. The hDus2 subfamily is proposed to specifically modify U20. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel beta-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain-domain interactions
evolution
comparison of structure and substrate recognition mechanisms of Thermus thermophilus Dus and Escherichia coli Dus enzymes, overview
evolution
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while in Dus enzymes from bacteria, plants and fungi, tRNA binding is essentially achieved by the alpha-helical domain, in HsDus2 this function is carried out by the dsRNA binding domain (dsRBD)
malfunction
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a small interfering RNA against hDUS2 transfected into NSCLC cells suppresses expression of the gene, reduces the amount of dihydrouridine in tRNA molecules, and suppresses growth
malfunction
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yeast extract from a dus1-DELTA strain is completely defective in modification of yeast pre-tRNAPhe
malfunction
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increased expression of human dihydrouridine synthase 2 (hDus2) is linked to pulmonary carcinogenesis, while its knockdown decreases cancer cell line viability
physiological function
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formation of dihydrouridine in yeast cytoplasmic tRNAs is carried out by a family of four dihydrouridine synthases (Dus1p, Dus2p, Dus3p, and Dus4p), each acting at specific positions in tRNAs. Dus1p modifies U16 and U17, Dus2p modifies U20, Dus3p modifies U47, and Dus4p modifies U20a and U20b. These four proteins are responsible for all dihydrouridine modification of cytoplasmic tRNAs in yeast
physiological function
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dihydrouridine is produced by dihydrouridine synthase (Dus) by enzymatic reduction of the C5-C6 bond in uridine
physiological function
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in tRNA, dihydrouridine is a conserved modified base generated by the post-transcriptional reduction of uridine. Formation of dihydrouridine 20, located in the D-loop, is catalyzed by dihydrouridine synthase 2 (Dus2). Full-length HsDus2 but not its Dus domain complements a DELTAdus2 yeast strain by catalyzing formation of dihydrouridine20
additional information
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residues that participate in binding to the adapter molecule in EcoDusC are Asn95, Lys139, Arg141, His168 and Arg170. The catalytic cysteine residue is Cys98
additional information
residues that participate in binding to the adapter molecule in EcoDusC are Asn95, Lys139, Arg141, His168 and Arg170. The catalytic cysteine residue is Cys98
additional information
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the catalytic domain binds selectively NADPH but cannot reduce uridine in the absence of the dsRNA binding domain (dsRBD). HsDus2 catalytic domain structure, overview
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
uracil in pre-tRNALeu + NAD(P)H + H+
5,6-dihydrouracil in pre-tRNALeu + NAD(P)+
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?
uracil in pre-tRNATyr + NAD(P)H + H+
5,6-dihydrouracil in pre-tRNATyr + NAD(P)+
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?
uracil in tRNA + NAD(P)H + H+
5,6-dihydrouracil in tRNA + NAD(P)+
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?
uracil in tRNA + NAD(P)H + H+
5,6-dihydrouracil in tRNA + NAD(P)+
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the enzyme is specific for the proR hydrogen of NADPH. Cys117 is very important for the reduction of tRNA
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r
uracil20 in tRNA + NAD(P)H + H+
5,6-dihydrouracil20 in tRNA + NAD(P)+
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?
uracil20 in tRNA + NAD(P)H + H+
5,6-dihydrouracil20 in tRNA + NAD(P)+
the enzyme specifically modifies uracil20 in tRNA, e.g. tRNALeu(CAA) and tRNATyr
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?
uracil20 in tRNA + NAD(P)H + H+
5,6-dihydrouracil20 in tRNA + NAD(P)+
e.g. tRNALeu(CAA) and tRNATyr
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?
additional information
?
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tRNA recognition mechanism, overview
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
uracil in tRNA + NAD(P)H + H+
5,6-dihydrouracil in tRNA + NAD(P)+
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?
uracil20 in tRNA + NAD(P)H + H+
5,6-dihydrouracil20 in tRNA + NAD(P)+
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?
uracil20 in tRNA + NAD(P)H + H+
5,6-dihydrouracil20 in tRNA + NAD(P)+
P53720
the enzyme specifically modifies uracil20 in tRNA, e.g. tRNALeu(CAA) and tRNATyr
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?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
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
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
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
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
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overexpression of hDUS2 in non–small cell lung carcinomas is identified by analysis of the gene-expression profiles
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
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the enzyme is composed of two domains: an N-terminal catalytic domain and a C-terminal tRNA-binding domain
additional information
the enzyme is composed of two domains: an N-terminal catalytic domain and a C-terminal tRNA-binding domain
additional information
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three-dimensional structure analysis of wild-type and selenomethionine-labeled hDus2 catalytic and tRNA-recognition domains, and modeling, overview
additional information
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the N-terminal catalytic domain contains the flavin cofactor involved in the reduction of uridine. The second module is the conserved alpha-helical domain known as the tRNA binding domain in HsDus2 homologues. It is connected via a flexible linker to an unusual extended version of a dsRNA binding domain (dsRBD). The catalytic domain binds selectively NADPH but cannot reduce uridine in the absence of the dsRBD. Domain architecture of HsDus2, enzyme MALDI peptide mass finger printing analysis, overview
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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 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. 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
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
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
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
medicine
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upregulation of hDUS2 is a relatively common feature of pulmonary carcinogenesis. Selective suppression of hDUS2 enzyme activity and/or inhibition of formation of the hDUS2-tRNA synthetase complex could be a promising therapeutic strategy for treatment of many lung cancers. Significant association between higher levels of hDUS2 in tumors and poorer prognosis of lung cancer patients
medicine
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the enzyme may serve as a valuable target for therapeutic intervention in pulmonary carcinogenesis
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