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
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)
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
increased expression of human dihydrouridine synthase 2 (hDus2) is linked to pulmonary carcinogenesis, while its knockdown decreases cancer cell line viability
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
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
residues that participate in binding to the adapter molecule in EcoDusC are Asn95, Lys139, Arg141, His168 and Arg170. The catalytic cysteine residue is Cys98
residues that participate in binding to the adapter molecule in EcoDusC are Asn95, Lys139, Arg141, His168 and Arg170. The catalytic cysteine residue is Cys98
the catalytic domain binds selectively NADPH but cannot reduce uridine in the absence of the dsRNA binding domain (dsRBD). HsDus2 catalytic domain structure, overview
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
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 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
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
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
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
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
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