scholarly journals Baculovirus expression of the 11 mycoreovirus-1 genome segments and identification of the guanylyltransferase-encoding segment

2007 ◽  
Vol 88 (1) ◽  
pp. 342-350 ◽  
Author(s):  
S. Supyani ◽  
Bradley I. Hillman ◽  
Nobuhiro Suzuki
Keyword(s):  
Active Site ◽  
Sequence Similarity ◽  
Expression System ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Sequence Alignments ◽  
High Sequence Similarity ◽  
Chestnut Blight Fungus ◽  
A Genome ◽  
Hypovirulent Strain

The type member Mycoreovirus 1 (MyRV-1) of a newly described genus, Mycoreovirus, isolated from a hypovirulent strain 9B21 of the chestnut blight fungus, has a genome composed of 11 dsRNA segments (S1–S11). All of the segments have single ORFs on their capped, positive-sense strands. Infection of insect cells by baculovirus recombinants carrying full-length cDNAs of S1–S11 resulted in overexpression of protein products of the expected sizes, based on their deduced amino acid sequences. This expression system was utilized to identify the S3-encoded protein (VP3) as a guanylyltransferase by an autoguanylylation assay, in which only VP3 was radiolabelled with [α-32P]GTP. A series of progressive N-terminal and C-terminal deletion mutants was made to localize the autoguanylylation-active site of VP3 to aa residues 133–667. Within this region, a sequence stretch (aa 170–250) with relatively high sequence similarity to homologues of two other mycoreoviruses and two coltiviruses was identified. Site-directed mutagenesis of conserved aa residues revealed that H233, H242, Y243, F244 and F246, but not K172 or K202, play critical roles in guanylyltransferase activities. Together with broader sequence alignments of ‘turreted’ reoviruses, these results supported the a/vxxHx8Hyf/lvf motif, originally noted for orthoreovirus and aquareoviruses, as an active site for guanylyltransferases of viruses within the Orthoreovirus, Aquareovirus, Cypovirus, Oryzavirus, Fijivirus, Coltivirus and Mycoreovirus genera, as well as for the proposed Dinovernavirus genus.

scholarly journals Different Roles of DosS and DosT in the Hypoxic Adaptation of Mycobacteria

2010 ◽  
Vol 192 (19) ◽  
pp. 4868-4875 ◽  
Author(s):  
Min-Ju Kim ◽  
Kwang-Jin Park ◽  
In-Jeong Ko ◽  
Young Min Kim ◽  
Jeong-Il Oh
Keyword(s):  
Oxygen Tension ◽  
Response Regulator ◽  
Sequence Similarity ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
High Sequence Similarity ◽  
Hypoxic Adaptation ◽  
The Difference ◽  
A Domains

ABSTRACT The DosS (DevS) and DosT histidine kinases form a two-component system together with the DosR (DevR) response regulator in Mycobacterium tuberculosis. DosS and DosT, which have high sequence similarity to each other over the length of their amino acid sequences, contain two GAF domains (GAF-A and GAF-B) in their N-terminal sensory domains. Complementation tests in conjunction with phylogenetic analysis showed that DevS of Mycobacterium smegmatis is more closely related to DosT than DosS. We also demonstrated in vivo that DosS and DosT of M. tuberculosis play a differential role in hypoxic adaptation. DosT responds to a decrease in oxygen tension more sensitively and strongly than DosS, which might be attributable to their different autooxidation rates. The different responsiveness of DosS and DosT to hypoxia is due to the difference in their GAF-A domains accommodating the hemes. Multiple alignment analysis of the GAF-A domains of mycobacterial DosS (DosT) homologs and subsequent site-directed mutagenesis revealed that just one substitution of E87, D90, H97, L118, or T169 of DosS with the corresponding residue of DosT is sufficient to convert DosS to DosT with regard to the responsiveness to changes in oxygen tension.

scholarly journals Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

2001 ◽  
Vol 67 (2) ◽  
pp. 673-679 ◽  
Author(s):  
Kazuhiko Ishikawa ◽  
Hiroyasu Ishida ◽  
Ikuo Matsui ◽  
Yutaka Kawarabayasi ◽  
Hisasi Kikuchi
Keyword(s):  
Active Site ◽  
Sequence Similarity ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Vector System ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
High Sequence Similarity ◽  
Pyrococcus Horikoshii ◽  
Thermophilic Archaeon

ABSTRACT Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase ofSulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus. The gene from P. horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90°C, with the highest activity above 95°C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.

Identification of the active site and characterization of a novel sporulation-specific cysteine protease YabG from Bacillus subtilis

2021 ◽  
Author(s):  
Ryuji Yamazawa ◽  
Ritsuko Kuwana ◽  
Kenji Takeuchi ◽  
Hiromu Takamatsu ◽  
Yoshitaka Nakajima ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Chemical Modification ◽  
Cysteine Protease ◽  
Active Site ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Protease Gene ◽  
Active Site Residues ◽  
Protein Determination ◽  
35 Kda Protein

Abstract In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) in Escherichia coli cells. During purification using Ni-affinity chromatography, the 35 kDa protein was degraded via several intermediates to form a 24 kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35 kDa protein to the 24 kDa protein was investigated. Reagents reacting with sulfhydryl groups exerted significant effects, strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibited the degradation of the 24 kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the Merops peptidase database.

scholarly journals N-Linked Glycosylation Modulates Golgi-Independent Vacuolar Sorting Mediated by the Plant Specific Insert

Plants ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 312 ◽  
Author(s):  
Vanessa Vieira ◽  
Bruno Peixoto ◽  
Mónica Costa ◽  
Susana Pereira ◽  
José Pissarra ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Site Directed Mutagenesis ◽  
Research Gaps ◽  
Glycosylation Pattern ◽  
High Sequence Similarity ◽  
Post Translational Modifications ◽  
Vacuolar Sorting ◽  
Prevacuolar Compartment ◽  
Cardosin A ◽  
Comprehensive Study

In plant cells, the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant-Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSI domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlike its native glycosylated form, is able to bypass ER-to-Golgi blockage and accumulate in the vacuole. This is also true when the PSI domain is analyzed in the context of the full-length cardosin. Regardless of opening exciting research gaps, the results obtained so far need a more comprehensive study of the mechanisms behind this unconventional direct sorting to the vacuole.

The vsp Locus of Mycoplasma bovis: Gene Organization and Structural Features

1999 ◽  
Vol 181 (18) ◽  
pp. 5734-5741 ◽  
Author(s):  
Inessa Lysnyansky ◽  
Konrad Sachse ◽  
Ricardo Rosenbusch ◽  
Sharon Levisohn ◽  
David Yogev
Keyword(s):  
High Frequency ◽  
Sequence Similarity ◽  
Membrane Surface ◽  
Amino Acid Sequences ◽  
High Rate ◽  
Phenotypic Switching ◽  
Upstream Region ◽  
Mycoplasma Bovis ◽  
High Sequence Similarity ◽  
Polypeptide Structure

ABSTRACT Major lipoprotein antigens, known as variable membrane surface lipoproteins (Vsps), on the surface of the bovine pathogenMycoplasma bovis were shown to spontaneously undergo noncoordinate phase variation between ON and OFF expression states. The high rate of Vsp phenotypic switching was also shown to be linked with DNA rearrangements that occur at high frequency in the M. bovis chromosome (I. Lysnyansky, R. Rosengarten, and D. Yogev, J. Bacteriol. 178:5395–5401, 1996). In the present study, 13 single-copyvsp genes organized in a chromosomal cluster were identified and characterized. All vsp genes encode highly conserved N-terminal domains for membrane insertion and lipoprotein processing but divergent mature Vsp proteins. About 80% of eachvsp coding region is composed of reiterated coding sequences that create a periodic polypeptide structure. Eighteen distinct repetitive domains of different lengths and amino acid sequences are distributed within the products of the variousvsp genes that are subject to size variation due to spontaneous insertions or deletions of these periodic units. Some of these repeats were found to be present in only one Vsp family member, whereas other repeats recurred at variable locations in several Vsps. Each vsp gene is also 5′ linked to a highly homologous upstream region composed of two internal cassettes. The findings that rearrangement events are associated with Vsp phenotypic switching and that multiple regions of high sequence similarity are present upstream of the vsp genes and within the vsp coding regions suggest that modulation of the Vsp antigenic repertoire is determined by recombination processes that occur at a high frequency within the vsp locus of M. bovis.

Caldariomyces fumago DSM1256 Contains Two Chloroperoxidase Genes, Both Encoding Secreted and Active Enzymes

2015 ◽  
Vol 25 (4) ◽  
pp. 237-243 ◽  
Author(s):  
Markus Buchhaupt ◽  
Sonja Hüttmann ◽  
Christian Carsten Sachs ◽  
Sebastian Bormann ◽  
Achim Hannappel ◽  
...  
Keyword(s):  
Active Site ◽  
Model Comparison ◽  
Catalytic Properties ◽  
Sequence Similarity ◽  
Structure Model ◽  
Transcriptome Data ◽  
High Sequence Similarity ◽  
Caldariomyces Fumago ◽  
C Terminus ◽  
High Conservation

Inspection of transcriptome data from the chloroperoxidase (CPO)-producing fungus <i>Caldariomyces fumago</i> DSM1256 led to the discovery of two distinct <i>CPO</i> mRNA sequences. This strain could be shown to contain the newly identified isogene as well as produce and secrete both isoenzymes. The CPO2 enzyme bears high sequence similarity to the well-characterized CPO (87% identity for the mature proteins). It shows two insertions in the signal peptide and in the C-terminal propeptide, and one deletion in the mature polypeptide close to the C-terminus. Furthermore, it lacks one of the serine residues known to be O-glycosylated in the CPO sequence. The demonstration of a <i>CPO</i> isogene which is expressed as a secreted and active CPO clarifies the nature of this isoenzyme already identified in earlier reports. A structure model comparison shows a high conservation of the active site and the substrate channel, suggesting very similar catalytic properties.

scholarly journals Molecular and Symptom Analysis Reveal the Presence of New Phytoplasmas Associated with Sugarcane Grassy Shoot Disease in India

Plant Disease ◽  
2007 ◽  
Vol 91 (11) ◽  
pp. 1413-1418 ◽  
Author(s):  
Kanchan Nasare ◽  
Amit Yadav ◽  
Anil K. Singh ◽  
K. B. Shivasharanappa ◽  
Y. S. Nerkar ◽  
...  
Keyword(s):  
16S Rrna ◽  
Sequence Similarity ◽  
Pcr Amplification ◽  
Saccharum Officinarum ◽  
23S Rrna ◽  
Rrna Gene ◽  
Sequence Alignments ◽  
High Sequence Similarity ◽  
Multiple Sequence ◽  
Very High

A total of 240 sugarcane (Saccharum officinarum) plants showing phenotypic symptoms of sugarcane grassy shoot (SCGS) disease were collected from three states of India, Maharashtra, Karnataka, and Uttar Pradesh. Phytoplasmas were detected in all symptomatic samples by the polymerase chain reaction (PCR) amplification of phytoplasma-specific 16S rRNA gene and 16S-23S rRNA spacer region (SR) sequences. No amplification was observed when DNA from asymptomatic plant samples was used as a template. Sixteen samples were selected on the basis of phenotypic symptoms and geographic location, and cloning and sequencing of the 16S rRNA and spacer regions were performed. Multiple sequence alignments of the 16S rRNA sequences revealed that they share very high sequence similarity with phytoplasmas of rice yellow dwarf, 16SrXI. However, the 16S-23S rRNA SR sequence analysis revealed that while the majority of phytoplasmas shared very high (>99%) sequence similarity with previously reported sugarcane phytoplasmas, two of them, namely BV2 (DQ380342) and VD7 (DQ380343), shared relatively low sequence similarity (79 and 84%, respectively). Therefore, these two phytoplasmas may be previously unreported ones that cause significant yield losses in sugarcane in India.

scholarly journals Characterization of Novel Erwinia amylovora Jumbo Bacteriophages from Eneladusvirus Genus

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1373
Author(s):  
Sang Guen Kim ◽  
Sung Bin Lee ◽  
Sib Sankar Giri ◽  
Hyoun Joong Kim ◽  
Sang Wha Kim ◽  
...  
Keyword(s):  
Genome Size ◽  
Sequence Similarity ◽  
Phylogenetic Analyses ◽  
Gc Content ◽  
Genomic Analysis ◽  
Open Reading Frames ◽  
Comparative Genomic ◽  
Limited Information ◽  
High Sequence Similarity ◽  
A Genome

Jumbo phages, which have a genome size of more than 200 kb, have recently been reported for the first time. However, limited information is available regarding their characteristics because few jumbo phages have been isolated. Therefore, in this study, we aimed to isolate and characterize other jumbo phages. We performed comparative genomic analysis of three Erwinia phages (pEa_SNUABM_12, pEa_SNUABM_47, and pEa_SNUABM_50), each of which had a genome size of approximately 360 kb (32.5% GC content). These phages were predicted to harbor 546, 540, and 540 open reading frames with 32, 34, and 35 tRNAs, respectively. Almost all of the genes in these phages could not be functionally annotated but showed high sequence similarity with genes encoded in Serratia phage BF, a member of Eneladusvirus. The detailed comparative and phylogenetic analyses presented in this study contribute to our understanding of the diversity and evolution of Erwinia phage and the genus Eneladusvirus.

Three Dimensional Computer Graphics Models Of Bovine Factor Xa, Factor IXa And Thrombin

1981 ◽  
Author(s):  
B Furie ◽  
D H Bing ◽  
B C Furie ◽  
D J Robison ◽  
J P Burnier ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Sequence Homology ◽  
Active Site ◽  
Three Dimensional ◽  
Factor Xa ◽  
Amino Acid Sequences ◽  
Molecular Surface ◽  
Sequence Alignments ◽  
Factor Ixa ◽  
Backbone Structure

Three dimensional structural models of the heavy chain of Factor Xa, the heavy chain of Factor IXa, and the B chain of thrombin have been developed using a computer molecular graphics display system. These models are based upon the sequence homology of these proteins with chymotrypsin and trypsin and the assumption that the three dimensional structures of the peptide backbones of these proteins are nearly identical to the known backbone structure of trypsin and chymotrypsin. Sequence alignments of each protein with the digestive proteases were based upon regions of sequence homology, location of disulfide bonds, and preservation of β-barrels. Regions of insertion and deletion relative to the chymotrypsin backbone were identified and incorporated into the backbone structure. Factor Xa (heavy chain) and Factor IXa (heavy chain) contain 5 insertions and 4 deletions. Thrombin (B chain) contains nine insertions and two deletions. Factor Xa, Factor IXa, and thrombin models were generated by substituting their respective amino acid sequences into the model of the peptide backbone. The molecular surfaces of these models each suggest unique topographical and physicochemical properties. In-contrast, the core of these proteins are nearly identical. One area of the molecular surface, the active site, is also highly conserved despite marked differences in substrate specificity among these enzymes. The extended substrate binding regions, surrounding the active site, are highly substituted and also contain areas of both insertions and deletions. These models predict that the functional differences that distinguish these serine proteases are manifestations of the molecular surface differences. It would appear that substrate recognition and specificity determinants of Factor Xa, Factor IXa, and thrombin reside in an extensive surface surrounding the active site, but not exclusively within the active site.

scholarly journals An Unusual Route for p-Aminobenzoate Biosynthesis in Chlamydia trachomatis Involves a Probable Self-Sacrificing Diiron Oxygenase

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Yamilet Macias-Orihuela ◽  
Thomas Cast ◽  
Ian Crawford ◽  
Kevin J. Brandecker ◽  
Jennifer J. Thiaville ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Active Site ◽  
De Novo ◽  
Sequence Similarity ◽  
Single Gene ◽  
The United States ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
Active Site Tyrosine

ABSTRACT Chlamydia trachomatis lacks the canonical genes required for the biosynthesis of p-aminobenzoate (pABA), a component of essential folate cofactors. Previous studies revealed a single gene from C. trachomatis, the CT610 gene, that rescues Escherichia coli ΔpabA, ΔpabB, and ΔpabC mutants, which are otherwise auxotrophic for pABA. CT610 shares low sequence similarity to nonheme diiron oxygenases, and the previously solved crystal structure revealed a diiron active site. Genetic studies ruled out several potential substrates for CT610-dependent pABA biosynthesis, including chorismate and other shikimate pathway intermediates, leaving the actual precursor(s) unknown. Here, we supplied isotopically labeled potential precursors to E. coli ΔpabA cells expressing CT610 and found that the aromatic portion of tyrosine was highly incorporated into pABA, indicating that tyrosine is a precursor for CT610-dependent pABA biosynthesis. Additionally, in vitro enzymatic experiments revealed that purified CT610 exhibits low pABA synthesis activity under aerobic conditions in the absence of tyrosine or other potential substrates, where only the addition of a reducing agent such as dithiothreitol appears to stimulate pABA production. Furthermore, site-directed mutagenesis studies revealed that two conserved active site tyrosine residues are essential for the pABA synthesis reaction in vitro. Thus, the current data are most consistent with CT610 being a unique self-sacrificing enzyme that utilizes its own active site tyrosine residue(s) for pABA biosynthesis in a reaction that requires O2 and a reduced diiron cofactor. IMPORTANCE Chlamydia trachomatis is the most reported sexually transmitted infection in the United States and the leading cause of infectious blindness worldwide. Unlike many other intracellular pathogens that have undergone reductive evolution, C. trachomatis is capable of de novo biosynthesis of the essential cofactor tetrahydrofolate using a noncanonical pathway. Here, we identify the biosynthetic precursor to the p-aminobenzoate (pABA) portion of folate in a process that requires the CT610 enzyme from C. trachomatis. We further provide evidence that CT610 is a self-sacrificing or “suicide” enzyme that uses its own amino acid residue(s) as the substrate for pABA synthesis. This work provides the foundation for future investigation of this chlamydial pABA synthase, which could lead to new therapeutic strategies for C. trachomatis infections.

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