scholarly journals Structural and functional characterization of the severe fever with thrombocytopenia syndrome virus L protein

2020 ◽  
Vol 48 (10) ◽  
pp. 5749-5765 ◽  
Author(s):  
Dominik Vogel ◽  
Sigurdur Rafn Thorkelsson ◽  
Emmanuelle R J Quemin ◽  
Kristina Meier ◽  
Tomas Kouba ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Functional Characterization ◽  
Limiting Factor ◽  
Genome Replication ◽  
Human Pathogens ◽  
Emerging Viruses ◽  
Replication Process ◽  
L Protein ◽  
Severe Fever

Abstract The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, is an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron cryo-microscopy and X-ray crystallography, we obtained 3D models covering ∼70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein–RNA interactions and the development of antiviral strategies against this group of emerging human pathogens.

scholarly journals Structural and functional characterization of the Severe fever with thrombocytopenia syndrome virus L protein

2020 ◽  
Author(s):  
Dominik Vogel ◽  
Sigurdur Rafn Thorkelsson ◽  
Emmanuelle R. J. Quemin ◽  
Kristina Meier ◽  
Tomas Kouba ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Functional Characterization ◽  
Limiting Factor ◽  
Genome Replication ◽  
Human Pathogens ◽  
Emerging Viruses ◽  
Replication Process ◽  
L Protein ◽  
Severe Fever

ABSTRACTThe Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, would be an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron-cryo microscopy and X-ray crystallography, we obtained 3D models covering ∼70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein-RNA interactions and the development of antiviral strategies against this group of emerging human pathogens.

scholarly journals Molecular and biochemical characterization of an endo-β-1,3-glucanase from the pinewood nematode Bursaphelenchus xylophilus acquired by horizontal gene transfer from bacteria

2005 ◽  
Vol 389 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Taisei KIKUCHI ◽  
Hajime SHIBUYA ◽  
John T. JONES
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Biochemical Characterization ◽  
Glycosyl Hydrolase ◽  
Functional Characterization ◽  
Nematode Species ◽  
Bursaphelenchus Xylophilus ◽  
Pinewood Nematode ◽  
Sequence Comparisons

We report the cloning and functional characterization of an endo-β-1,3-glucanase from the pinewood nematode Bursaphelenchus xylophilus acquired by horizontal gene transfer from bacteria. This is the first gene of this type from any nematode species. We show that a similar cDNA is also present in another closely related species B. mucronatus, but that similar sequences are not present in any other nematode studied to date. The B. xylophilus gene is expressed solely in the oesophageal gland cells of the nematode and the protein is present in the nematode's secretions. The deduced amino acid sequence of the gene is very similar to glycosyl hydrolase family 16 proteins. The recombinant protein, expressed in Escherichia coli, preferentially hydrolysed the β-1,3-glucan laminarin, and had very low levels of activity on β-1,3-1,4-glucan, lichenan and barley β-glucan. Laminarin was degraded in an endoglucanase mode by the enzyme. The optimal temperature and pH for activity of the recombinant enzyme were 65 °C and pH 4.9. The protein is probably important in allowing the nematodes to feed on fungi. Sequence comparisons suggest that the gene encoding the endo-β-1,3-glucanase was acquired by horizontal gene transfer from bacteria. B. xylophilus therefore contains genes that have been acquired by this process from both bacteria and fungi. These findings support the idea that multiple independent horizontal gene transfer events have helped in shaping the evolution of several different life strategies in nematodes.

scholarly journals Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

2020 ◽  
Vol 75 (9) ◽  
pp. 2554-2563 ◽  
Author(s):  
Christopher Fröhlich ◽  
Vidar Sørum ◽  
Sandra Huber ◽  
Ørjan Samuelsen ◽  
Fanny Berglund ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Human Pathogens ◽  
E Coli ◽  
X Ray Crystallography ◽  
Environmental Reservoir

Abstract Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). Results Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. Conclusions These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

scholarly journals RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

2009 ◽  
Vol 90 (7) ◽  
pp. 1748-1756 ◽  
Author(s):  
M. Gopinath ◽  
M. S. Shaila
Keyword(s):  
Biochemical Characterization ◽  
Covalent Bond ◽  
Rinderpest Virus ◽  
Viral Mrnas ◽  
L Protein ◽  
Rna Triphosphatase ◽  
Rnp Complex

Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L–P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of γ-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 5′ sequence. The L protein forms a covalent enzyme–guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717–2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.

scholarly journals Functional characterization of heat-shock protein 90 fromOryza sativaand crystal structure of its N-terminal domain

2015 ◽  
Vol 71 (6) ◽  
pp. 688-696 ◽  
Author(s):  
Swetha Raman ◽  
Kaza Suguna
Keyword(s):  
Crystal Structure ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Biochemical Characterization ◽  
Functional Characterization ◽  
Cell Signalling ◽  
Physiological Role ◽  
Heat Shock Protein 90 ◽  
Terminal Domain

Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 Å resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 fromDictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source.

scholarly journals Isolation and characterization of two O-methyltransferases involved in benzylisoquinoline alkaloid biosynthesis in sacred lotus (Nelumbo nucifera)

2019 ◽  
Vol 295 (6) ◽  
pp. 1598-1612 ◽  
Author(s):  
Ivette M. Menéndez-Perdomo ◽  
Peter J. Facchini
Keyword(s):  
Biochemical Characterization ◽  
Functional Characterization ◽  
Nelumbo Nucifera ◽  
Plant Metabolites ◽  
Sacred Lotus ◽  
Isolation And Characterization ◽  
Medicinal Value ◽  
Benzylisoquinoline Alkaloid ◽  
Young Leaves

Benzylisoquinoline alkaloids (BIAs) are a major class of plant metabolites with many pharmacological benefits. Sacred lotus (Nelumbo nucifera) is an ancient aquatic plant of medicinal value because of antiviral and immunomodulatory activities linked to its constituent BIAs. Although more than 30 BIAs belonging to the 1-benzylisoquinoline, aporphine, and bisbenzylisoquinoline structural subclasses and displaying a predominant R-enantiomeric conformation have been isolated from N. nucifera, its BIA biosynthetic genes and enzymes remain unknown. Herein, we report the isolation and biochemical characterization of two O-methyltransferases (OMTs) involved in BIA biosynthesis in sacred lotus. Five homologous genes, designated NnOMT1–5 and encoding polypeptides sharing >40% amino acid sequence identity, were expressed in Escherichia coli. Functional characterization of the purified recombinant proteins revealed that NnOMT1 is a regiospecific 1-benzylisoquinoline 6-O-methyltransferase (6OMT) accepting both R- and S-substrates, whereas NnOMT5 is mainly a 7-O-methyltransferase (7OMT), with relatively minor 6OMT activity and a strong stereospecific preference for S-enantiomers. Available aporphines were not accepted as substrates by either enzyme, suggesting that O-methylation precedes BIA formation from 1-benzylisoquinoline intermediates. Km values for NnOMT1 and NnOMT5 were 20 and 13 μm for (R,S)-norcoclaurine and (S)-N-methylcoclaurine, respectively, similar to those for OMTs from other BIA-producing plants. Organ-based correlations of alkaloid content, OMT activity in crude extracts, and OMT gene expression supported physiological roles for NnOMT1 and NnOMT5 in BIA metabolism, occurring primarily in young leaves and embryos of sacred lotus. In summary, our work identifies two OMTs involved in BIA metabolism in the medicinal plant N. nucifera.

scholarly journals Comparative Biochemical Characterization of Three Exolytic Oligoalginate Lyases from Vibrio splendidus Reveals Complementary Substrate Scope, Temperature, and pH Adaptations

2014 ◽  
Vol 80 (14) ◽  
pp. 4207-4214 ◽  
Author(s):  
Sujit Sadashiv Jagtap ◽  
Jan-Hendrik Hehemann ◽  
Martin F. Polz ◽  
Jung-Kul Lee ◽  
Huimin Zhao
Keyword(s):  
Biochemical Characterization ◽  
Functional Characterization ◽  
Catalytic Efficiency ◽  
Vibrio Splendidus ◽  
Marine Microbes ◽  
Ph Optimum ◽  
Brown Seaweeds ◽  
Content Type ◽  
Alginate Lyases

ABSTRACTMarine microbes use alginate lyases to degrade and catabolize alginate, a major cell wall matrix polysaccharide of brown seaweeds. Microbes frequently contain multiple, apparently redundant alginate lyases, raising the question of whether these enzymes have complementary functions. We report here on the molecular cloning and functional characterization of three exo-type oligoalginate lyases (OalA, OalB, and OalC) fromVibrio splendidus12B01 (12B01), a marine bacterioplankton species. OalA was most active at 16°C, had a pH optimum of 6.5, and displayed activities toward poly-β-d-mannuronate [poly(M)] and poly-α-l-guluronate [poly(G)], indicating that it is a bifunctional enzyme. OalB and OalC were most active at 30 and 35°C, had pH optima of 7.0 and 7.5, and degraded poly(M·G) and poly(M), respectively. Detailed kinetic analyses of oligoalginate lyases with poly(G), poly(M), and poly(M·G) and sodium alginate as substrates demonstrated that OalA and OalC preferred poly(M), whereas OalB preferred poly(M·G). The catalytic efficiency (kcat/Km) of OalA against poly(M) increased with decreasing size of the substrate. OalA showedkcat/Kmfrom 2,130 mg−1ml s−1for the trisaccharide to 224 mg−1ml s−1for larger oligomers of ∼50 residues, and 50.5 mg−1ml s−1for high-molecular-weight alginate. Although OalA was most active on the trisaccharide, OalB and OalC preferred dimers. Taken together, our results indicate that these three Oals have complementary substrate scopes and temperature and pH adaptations.

scholarly journals Structural and functional characterization of hemp seed (Cannabis sativa L.) protein-derived antioxidant and antihypertensive peptides

2014 ◽  
Vol 6 ◽  
pp. 384-394 ◽  
Author(s):  
Abraham T. Girgih ◽  
Rong He ◽  
Sunday Malomo ◽  
Marina Offengenden ◽  
Jianping Wu ◽  
...  
Keyword(s):  
Cannabis Sativa ◽  
Functional Characterization ◽  
Hemp Seed ◽  
L Protein ◽  
Antihypertensive Peptides

Rhizobium etli HrpW is a pectin-degrading enzyme and differs from phytopathogenic homologues in enzymically crucial tryptophan and glycine residues

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 3045-3054 ◽  
Author(s):  
Maarten Fauvart ◽  
Natalie Verstraeten ◽  
Bruno Dombrecht ◽  
Ruth Venmans ◽  
Serge Beullens ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Plant Cell Wall ◽  
Biochemical Characterization ◽  
Sequence Data ◽  
Functional Characterization ◽  
Pectate Lyase ◽  
Site Directed Mutagenesis ◽  
Rhizobium Etli ◽  
Lyase Activity

While establishing a nitrogen-fixing symbiosis with leguminous plants, rhizobia are faced with the problem of penetrating the plant cell wall at several stages of the infection process. One of the major components of this barrier is pectin, a heteropolysaccharide composed mainly of galacturonic acid subunits. So far, no enzymes capable of degrading pectin have been isolated from rhizobia. Here, we make an inventory of rhizobial candidate pectinolytic enzymes based on available genome sequence data and present an initial biochemical and functional characterization of a protein selected from this list. Rhizobium etli hrpW is associated with genes encoding a type III secretion system, a macromolecular structure that allows bacteria to directly inject so-called effector proteins into a eukaryotic host's cell cytosol and an essential virulence determinant of many Gram-negative pathogenic bacteria. In contrast to harpin HrpW from phytopathogens, R. etli HrpW possesses pectate lyase activity and is most active on highly methylated substrates. Through comparative sequence analysis, three amino acid residues crucial for the observed enzymic activity were identified: Trp192, Gly212 and Gly213. Their importance was confirmed by site-directed mutagenesis and biochemical characterization of the resulting proteins, with the tryptophan mutant showing no detectable pectate lyase activity and the double-glycine mutant's activity reduced by about 80 %. Surprisingly, despite hrpW expression being induced specifically on the plant root surface, a knockout mutation of the gene does not appear to affect symbiosis with the common bean Phaseolus vulgaris.

scholarly journals Analysis of Secreted Proteins and Potential Virulence via the ICEs-Mediated Pathway of the Foodborne Pathogen Vibrio parahaemolyticus

2021 ◽  
Vol 12 ◽  
Author(s):  
Yu He ◽  
Shuai Wang ◽  
Kaiwen Wang ◽  
Jinwei Zhou ◽  
Zhi Han ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Biochemical Characterization ◽  
Functional Characterization ◽  
Foodborne Pathogen ◽  
Secreted Proteins ◽  
Secretion Systems ◽  
Two Dimensional Gel Electrophoresis ◽  
Integrative And Conjugative Elements ◽  
The Relationship

Vibrio parahaemolyticus uses bacterial secretion systems and integrative and conjugative elements (ICEs) to induce various diseases and to adapt to harsh environments, respectively. Information pertaining to the identity of secreted proteins and functional characterization of ICEs has been previously reported, but the relationship between these elements remains unclear. Herein we investigated secreted proteins of V. parahaemolyticus strains JHY20 and JHY20△ICE using two-dimensional gel electrophoresis and LC-MS/MS, which led to the identification of an ICE-associated secreted protein – dihydrolipoamide dehydrogenase (DLDH). Considering the data related to its physical and biochemical characterization, we predicted that DLDH is a novel immunogenic protein and associated with virulence in JHY20. Our findings indicate a potential relationship between ICE-associated transport and secreted proteins and shed light on the function of such transport mechanisms. We believe that our data should enhance our understanding of mobile genetic elements.

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