scholarly journals Purification and Characterization of Double-Stranded Nucleic Acid-Dependent ATPase Activities of Tagged Dicer-Related Helicase 1 and its Short Isoform in Caenorhabditis elegans

Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 734
Author(s):  
Taishi Kobayashi ◽  
Takuro Murakami ◽  
Yuu Hirose ◽  
Toshihiko Eki
Keyword(s):  
Caenorhabditis Elegans ◽  
Nucleic Acid ◽  
Atpase Activity ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Sumo Protease ◽  
Dependent Atpase ◽  
Antiviral Function ◽  
Isoform B

The Dicer-related helicases (DRHs) are members of a helicase subfamily, and mammalian DRHs such as retinoic acid-inducible gene-I (RIG-I), are involved in antiviral immunity. Caenorhabditis elegans DRH-1 and DRH-3 play crucial roles in antiviral function and chromosome segregation, respectively. Although intrinsic double-stranded RNA-dependent ATP-hydrolyzing activity has been observed in the recombinant DRH-3 protein prepared from Escherichia coli, there are no reports of biochemical studies of the nematode RIG-I homolog DRH-1. In this study, the secondary structure prediction by JPred4 revealed that DRH-1 and DRH-3 had distinct N-terminal regions and that a 200-amino acid N-terminal region of DRH-1 could form a structure very rich in α-helices. We investigated expressions and purifications of a codon-optimized DRH-1 with four different N-terminal tags, identifying poly-histidine (His)-small ubiquitin-like modifier (SUMO) as a suitable tag for DRH-1 preparation. Full-length (isoform a) and a N-terminal truncated (isoform b) of DRH-1 were purified as the His-SUMO-tagged fusion proteins. Finally, the nucleic acid-dependent ATPase activities were investigated for the two His-SUMO-tagged DRH-1 isoforms and His-tagged DRH-3. The tagged DRH-3 exhibited dsRNA-dependent ATPase activity. However, detectable dsRNA dependency of ATPase activities was not found in either isoform of tagged DRH-1 and a tag-free DRH-1 (isoform a) treated with SUMO protease. These observations suggest that DRH-1 and its short isoform have no or poor nucleic acid-dependent ATPase activity, unlike DRH-3 and mammalian DRHs.

Characterization of the autoantigen La as a nucleic acid-dependent ATPase/dATPase with melting properties

Cell ◽  
1990 ◽  
Vol 60 (1) ◽  
pp. 85-93 ◽  
Author(s):  
Michael Bachmann ◽  
Karin Pfeifer ◽  
Heinz-C. Schrdder ◽  
Werner E.G. Müller
Keyword(s):  
Nucleic Acid ◽  
Dependent Atpase ◽  
Melting Properties

scholarly journals Molecular characterization of the viral structural protein genes of 2018 dengue virus serotype 1 epidemic in Yunnan, Southwest China

2020 ◽  
Author(s):  
Qingping Lan ◽  
Yun Shu ◽  
Linhao Li ◽  
Xiyun Shan ◽  
Dehong Ma ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Homologous Recombination ◽  
Molecular Characterization ◽  
Structure Prediction ◽  
Southwest China ◽  
Secondary Structure Prediction ◽  
Virus Serotype ◽  
Serotype 1 ◽  
Dengue Virus Serotype 1

Abstract Background There was a dengue virus serotype 1 (DENV-1) epidemic from October to December in 2018 in Xishuangbanna, Yunnan, southwest China, neighboring Myanmar, Laos and Vietnam. And the molecular characterization, evolution and potential source of DENV from Xishuangbanna were investigate. Methods C (capsid)/prM(premembrane)/E (envelope) genes of DNEV isolated from 87 serum samples obtained from local patients were amplified and sequenced, then evaluated using mutation, phylogenetic and homologous recombination analyses and secondary structure prediction. Results Phylogenetic analysis show that all the DENV strains from Xishuangbanna can grouped in two branch of DENV-1. Out of 52, 50 of isolates were grouped in branch one, and were most similar to Fujian (DQ193572) and Singapore 2016(MF314188) strains. The other two isolates have the closest relationship to Myanmar 2017 (MG679801) . When compared with DENV-1SS (standard strain), we found the amino acid residue of E155 was mutation from T to S,which may related to the virulence of virus and no obvious homologous recombination signals. Secondary structure prediction showed that some changes were found in the helical of MN123849 and MN123854 strains and few changes observed in disordered region. Conclusions This study revealed the molecular characterization of structural genes of xishuangbanna, 2018 epidemic strain, providing a reference for molecular epidemiology, infection, pathogenicity and vaccine development .

Cloning and Characterization of Three Genes Encoding LMW-GSs from Triticum aestivum, Cultival Cheyenne

2012 ◽  
Vol 554-556 ◽  
pp. 1116-1120 ◽  
Author(s):  
Mei Rong Chen ◽  
Xing Shen ◽  
Lin Li ◽  
Song Qing Hu
Keyword(s):  
Amino Acid ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Amino Acid Residues ◽  
Repetitive Domain ◽  
Genebank Accession ◽  
Genes Encoding ◽  
Α Helix ◽  
Β Sheet

Three low molecular weight subunit genes, named LMW-CND1 (GeneBank accession JQ780048), LMW-CND2 (GeneBank accession JQ779840), LMW-CND3 (GeneBank accession JQ779841), with a ORF of 1053 bp, 903 bp, 969 bp, respectively, were isolated from cv. Cheyenne and characterized detailed in molecular level. The proteins encoded by the genes, with 350, 300, 322 amino acid residues respectively, differ only in repetitive domain of sequences due to insertion or deletion of repeats in this domain. Highly similarity in amino-acid sequence between these three subunits and other published LMW-GSs was also observed, showing that all three genes published here are typical LMW-GS genes and closely related to the genes on chromosome 1D. Besides, secondary structure prediction of proteins indicated that, in the three LMW-GSs, random loop accounts for no less than 70 %, α-helix amounts to 26 %, average, and only 1.4 %~1.7 % is β-sheet.

scholarly journals Molecular and Mechanistic Characterization of PddB, the First PLP-Independent 2,4-Diaminobutyric Acid Racemase Discovered in an Actinobacterial D-Amino Acid Homopolymer Biosynthesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Kazuya Yamanaka ◽  
Ryo Ozaki ◽  
Yoshimitsu Hamano ◽  
Tadao Oikawa
Keyword(s):  
Amino Acid ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Sequence Similarity ◽  
Structural Difference ◽  
Structural Modeling ◽  
Specific Substrate ◽  
Amino Acid Residues ◽  
Diaminobutyric Acid

We recently disclosed that the biosynthesis of antiviral γ-poly-D-2,4-diaminobutyric acid (poly-D-Dab) in Streptoalloteichus hindustanus involves an unprecedented cofactor independent stereoinversion of Dab catalyzed by PddB, which shows weak homology to diaminopimelate epimerase (DapF). Enzymological properties and mechanistic details of this enzyme, however, had remained to be elucidated. Here, through a series of biochemical characterizations, structural modeling, and site-directed mutageneses, we fully illustrate the first Dab-specific PLP-independent racemase PddB and further provide an insight into its evolution. The activity of the recombinant PddB was shown to be optimal around pH 8.5, and its other fundamental properties resembled those of typical PLP-independent racemases/epimerases. The enzyme catalyzed Dab specific stereoinversion with a calculated equilibrium constant of nearly unity, demonstrating that the reaction catalyzed by PddB is indeed racemization. Its activity was inhibited upon incubation with sulfhydryl reagents, and the site-directed substitution of two putative catalytic Cys residues led to the abolishment of the activity. These observations provided critical evidence that PddB employs the thiolate-thiol pair to catalyze interconversion of Dab isomers. Despite the low levels of sequence similarity, a phylogenetic analysis of PddB indicated its particular relevance to DapF among PLP-independent racemases/epimerases. Secondary structure prediction and 3D structural modeling of PddB revealed its remarkable conformational analogy to DapF, which in turn allowed us to predict amino acid residues potentially responsible for the discrimination of structural difference between diaminopimelate and its specific substrate, Dab. Further, PddB homologs which seemed to be narrowly distributed only in actinobacterial kingdom were constantly encoded adjacent to the putative poly-D-Dab synthetase gene. These observations strongly suggested that PddB could have evolved from the primary metabolic DapF in order to organize the biosynthesis pathway for the particular secondary metabolite, poly-D-Dab. The present study is on the first molecular characterization of PLP-independent Dab racemase and provides insights that could contribute to further discovery of unprecedented PLP-independent racemases.

The glycomes of Caenorhabditis elegans and other model organisms

2002 ◽  
Vol 69 ◽  
pp. 117-134 ◽  
Author(s):  
Stuart M. Haslam ◽  
David Gems ◽  
Howard R. Morris ◽  
Anne Dell
Keyword(s):  
Caenorhabditis Elegans ◽  
Current Knowledge ◽  
Structural Data ◽  
Model Organisms ◽  
Entire Genome ◽  
C Elegans ◽  
The Face ◽  
Area Of Concern ◽  
Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

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