Cloning, Sequencing, and In Silico Analysis of β-Propeller Phytase Bacillus licheniformis Strain PB-13

β-Propeller phytases (BPPhy) are widely distributed in nature and play a major role in phytate-phosphorus cycling. In the present study, a BPPhy gene from Bacillus licheniformis strain was expressed in E. coli with a phytase activity of 1.15 U/mL and specific activity of 0.92 U/mg proteins. The expressed enzyme represented a full length ORF “PhyPB13” of 381 amino acid residues and differs by 3 residues from the closest similar existing BPPhy sequences. The PhyPB13 sequence was characterized in silico using various bioinformatic tools to better understand structural, functional, and evolutionary aspects of BPPhy class by multiple sequence alignment and homology search, phylogenetic tree construction, variation in biochemical features, and distribution of motifs and superfamilies. In all sequences, conserved sites were observed toward their N-terminus and C-terminus. Cysteine was not present in the sequence. Overall, three major clusters were observed in phylogenetic tree with variation in biophysical characteristics. A total of 10 motifs were reported with motif “1” observed in all 44 protein sequences and might be used for diversity and expression analysis of BPPhy enzymes. This study revealed important sequence features of BPPhy and pave a way for determining catalytic mechanism and selection of phytase with desirable characteristics.

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Comparative Study on Mitogen Activated Protein Kinase of Plasmodium Species by Using In silico Method

Scientific Research Journal ◽

10.24191/srj.v9i1.5052 ◽

2012 ◽

Vol 9 (1) ◽

pp. 1

Author(s):

Mohd Fakharul Zaman Raja Yahya ◽

Hasidah Mohd Sidek

Keyword(s):

Amino Acid ◽

In Silico ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Plasmodium Species ◽

Mitogen Activated Protein Kinase ◽

Multiple Sequence ◽

Bioinformatic Tools ◽

Wide Range ◽

Human Plasmodium

Malaria parasites, Plasmodium can infect a wide range ofhosts including humans and rodents. There are two copies ofmitogen activated protein kinases (MAPKs) in Plasmodium, namely MAPK1 and MAPK2. The MAPKs have been studied extensively in the human Plasmodium, P. falciparum. However, the MAPKs from other Plasmodium species have not been characterized and it is therefore the premise ofpresented study to characterize the MAPKs from other Plasmodium species-P. vivax, P. knowlesi, P. berghei, P. chabaudi and P.yoelli using a series ofpublicly available bioinformatic tools. In silico data indicates that all Plasmodium MAPKs are nuclear-localizedandcontain both a nuclear localization signal (NLS) anda Leucine-rich nuclear export signal (NES). The activation motifs ofTDYand TSH werefound to befully conserved in Plasmodium MAPK1 and MAPK2, respectively. The detailed manual inspection ofa multiple sequence alignment (MSA) construct revealed a total of 17 amino acid stack patterns comprising ofdifferent amino acids present in MAPK1 and MAPK2 respectively, with respect to rodent and human Plasmodia. 1t is proposed that these amino acid stack patterns may be useful in explaining the disparity between rodent and human Plasmodium MAPKs.

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In Silico Study of 1, 4 Alpha Glucan Branching Enzyme and Substrate Docking Studies

Current Proteomics ◽

10.2174/1570164616666190401204009 ◽

2020 ◽

Vol 17 (1) ◽

pp. 40-50

Author(s):

Farzane Kargar ◽

Amir Savardashtaki ◽

Mojtaba Mortazavi ◽

Masoud Torkzadeh Mahani ◽

Ali Mohammad Amani ◽

...

Keyword(s):

Phylogenetic Tree ◽

In Silico ◽

Alpha Helix ◽

In Silico Analysis ◽

Glycogen Storage ◽

Docking Studies ◽

Random Coil ◽

Special Topic ◽

Industrial Biotechnology ◽

In Silico Study

Background: The 1,4-alpha-glucan branching protein (GlgB) plays an important role in the glycogen biosynthesis and the deficiency in this enzyme has resulted in Glycogen storage disease and accumulation of an amylopectin-like polysaccharide. Consequently, this enzyme was considered a special topic in clinical and biotechnological research. One of the newly introduced GlgB belongs to the Neisseria sp. HMSC071A01 (Ref.Seq. WP_049335546). For in silico analysis, the 3D molecular modeling of this enzyme was conducted in the I-TASSER web server. Methods: For a better evaluation, the important characteristics of this enzyme such as functional properties, metabolic pathway and activity were investigated in the TargetP software. Additionally, the phylogenetic tree and secondary structure of this enzyme were studied by Mafft and Prabi software, respectively. Finally, the binding site properties (the maltoheptaose as substrate) were studied using the AutoDock Vina. Results: By drawing the phylogenetic tree, the closest species were the taxonomic group of Betaproteobacteria. The results showed that the structure of this enzyme had 34.45% of the alpha helix and 45.45% of the random coil. Our analysis predicted that this enzyme has a potential signal peptide in the protein sequence. Conclusion: By these analyses, a new understanding was developed related to the sequence and structure of this enzyme. Our findings can further be used in some fields of clinical and industrial biotechnology.

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The Drosophila DCP2 is evolutionarily conserved in sequence and structure : insights from in silico studies of DmDCP2 orthologs and paralogs

10.1101/2021.04.18.440350 ◽

2021 ◽

Author(s):

Rohit Kunar ◽

Jagat Kumar Roy

Keyword(s):

Phylogenetic Tree ◽

In Silico ◽

Evolutionary Relationship ◽

Structural Features ◽

Quantitative Approach ◽

Its Sequence ◽

Multiple Sequence ◽

Mrna Decapping ◽

In Silico Studies ◽

Evolutionarily Conserved

The mRNA decapping proteins (DCPs) function to hydrolyze the 7-methylguanosine cap at the 5 ′ end of mRNAs thereby, exposing the transcript for degradation by the exonuclease(s) and hence, play a pioneering role in the mRNA decay pathway. In Drosophila melanogaster, the mRNA decapping protein 2 (DCP2) is the only catalytically active mRNA decapping enzyme present. Despite its presence being reported across diverse species in the phylogenetic tree, a quantitative approach to the index of its conservation in terms of its sequence has not been reported so far. With structural and mechanistic insights being explored in the yeasts, the insect DCP2 has never been explored in the perspectives of structure and the indices of the conservation of its sequence and/or structure vis-a-vis topological facets. Being an evolutionarily conserved protein, the present endeavor aimed at deciphering the evolutionary relationship(s) and the pattern of conservation of the sequence of DCP2 across the phylogenetic tree as well as in sibling species of D. melanogaster through a semi-quantitative approach relying on multiple sequence alignment and analyses of percentage identity matrices. Since NUDIX proteins are functionally diverse, an attempt to identify the other NUDIX proteins (or, DCP2 paralogs) in D. melanogaster and compare and align their structural features with that of DCP2 through in silico approaches was endeavored in parallel. Our observations provide quantitative and structural bases for the observed evolutionary conservation of DCP2 across the diverse phyla and also, identify and reinforce the structural conservation of the NUDIX family in D. melanogaster.

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Novel Insights into the Existence of the Putative UDP-Glucuronate 5-Epimerase Specificity

Catalysts ◽

10.3390/catal10020222 ◽

2020 ◽

Vol 10 (2) ◽

pp. 222 ◽

Cited By ~ 3

Author(s):

Ophelia Gevaert ◽

Stevie Van Overtveldt ◽

Matthieu Da Costa ◽

Koen Beerens ◽

Tom Desmet

Keyword(s):

Phylogenetic Tree ◽

In Silico ◽

Molecular Level ◽

In Silico Analysis ◽

Current Work ◽

Skin Extract ◽

Rabbit Skin ◽

Uniprot Database ◽

Silico Analysis

C5-epimerases are promising tools for the production of rare l-hexoses from their more common d-counterparts. On that account, UDP-glucuronate 5-epimerase (UGA5E) attracts attention as this enzyme could prove to be useful for the synthesis of UDP-l-iduronate. Interestingly, l-iduronate is known as a precursor for the production of heparin, an effective anticoagulant. To date, the UGA5E specificity has only been detected in rabbit skin extract, and the respective enzyme has not been characterized in detail or even identified at the molecular level. Accordingly, the current work aimed to shed more light on the properties of UGA5E. Therefore, the pool of putative UGA5Es present in the UniProt database was scrutinized and their sequences were clustered in a phylogenetic tree. However, the examination of two of these enzymes revealed that they actually epimerize UDP-glucuronate at the 4- rather than 5-position. Furthermore, in silico analysis indicated that this should be the case for all sequences that are currently annotated as UGA5E and, hence, that such activity has not yet been discovered in nature. The detected l-iduronate synthesis in rabbit skin extract can probably be assigned to the enzyme chondroitin-glucuronate C5-epimerase, which catalyzes the conversion of d-glucuronate to l-iduronate on a polysaccharide level.

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Characterization of a Monoclonal Antibody, D73H, that Maps to a Highly Conserved Region on Fibrinogen Bβ Chain

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613965 ◽

2000 ◽

Vol 84 (07) ◽

pp. 43-48 ◽

Cited By ~ 2

Author(s):

B. J. Rybarczyk ◽

M. Pereira ◽

P. J. Simpson-Haidaris

Keyword(s):

In Silico ◽

Coiled Coil ◽

In Silico Analysis ◽

Antigenic Determinants ◽

Human Fibrinogen ◽

Western Blots ◽

C Terminus ◽

Probability Prediction ◽

Silico Analysis

SummaryThe primary structure of fibrinogen is highly conserved across species, yet often times monoclonal antibodies produced against the fibrinogen of one species will not crossreact with the fibrinogen of another. Herein, we describe the production and characterization of murine MAb, D73H, raised against human fibrinogen. D73H crossreacts with a highly conserved epitope on the Bβ chain of fibrinogen from human, rat, bovine, guinea pig, and mouse. Western blotting revealed that D73H reacted with the Bβ chain of plasmin fragment D, localizing its epitope to Bβ134-461. A 7 kDa band was identified by D73H in Western blots of reduced fibrinogen CNBr-fragments. N-terminal sequencing mapped this fragment to Bβ243-253, further localizing the epitope to Bβ243-305. In silico analysis indicated that Bβ243-305 is predominantly hydrophilic, and surface probability prediction indicated three potential antigenic determinants corresponding to Bβ252-258, Bβ262-269, and Bβ279-286. Further in silico analysis of the crystal structure of fibrinogen fragment D-D indicated that Bβ262-269 (FGRKWDPY) is predominantly α-helical and located on the surface of the molecule adjacent to a bend imposed in the β chain at residue 260, which is near the junction between the rigid coiled-coil domain and the globular C-terminus. A synthetic peptide corresponding to Bβ261-272 competitively inhibited the binding of D73H to the Bβ chain of denatured intact fibrinogen and reduced and denatured Bβ chain in Western blots, experimentally proving the validity of these predictive algorithms. Together these data indicate that, although plasmin resistant, Bβ chain residues Bβ261-272 comprising the D73H epitope are highly conserved across species, surface exposed, and immunogenic.

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The structural insight of class III of polyhydroxyalkanoate synthase from Bacillus sp. PSA10 as revealed by in silico analysis

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.53717 ◽

2020 ◽

Vol 25 (1) ◽

pp. 33

Author(s):

Listia Pradani ◽

Muhammad Saifur Rohman ◽

Sebastian Margino

Keyword(s):

In Silico ◽

Catalytic Mechanism ◽

In Silico Analysis ◽

Bacillus Sp ◽

Class Iii ◽

Reading Frame ◽

Structural Prediction ◽

Hydrolase Fold ◽

Sds Page ◽

Polyhydroxyalkanoate Synthase

PhaC synthase is an enzyme responsible for PHA polymerization. In this work, the catalytic mechanism class III of PhaC synthase from Bacillus sp. PSA10 (BacPhaCSynt) was reported through in silico modelling approach based on the primary sequence of the PhaC synthase. The open reading frame BacPhaCSynt has been successfully isolated, cloned and overexpressed the recombinant protein in Escherichia coli BL21(DE3). To know the global architecture and catalytic mechanism, the structural prediction of BacPhaCSynt has been carried out by using MODELLER. The recombinant BacPhaCSynt exhibited monomeric molecular weight (MW) of 43.6 kDa, when it was analyzed on 12% SDS‐PAGE gel. Based on the structural prediction, BacPhaCSynt exhibited global architecture of α/β hydrolase fold, with the root mean square deviation (r.m.s.d) value of 0.94Å. The catalytic residues composition of BacPhaCSynt consists of C151, D307, and H336, but the H336 and D307 residues of the model have been distorted 62.8o and 175.2o from the corresponding residues of the template. Since the D307 is quite a distance from the H336, it might act as a general base for the activation of ‐OH group of the substrate. The results strongly suggested that the mode of action of BacPhaCSynt obeyed the covalent catalysis mechanism.

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Genetics A Comprehensive In Silico Analysis of Deleterious SNPs of Paraplegin Protein Associated with Hereditary Spastic Paraplegia through Mitochondrial Dysfunction

BioScientific Review ◽

10.32350/bsr.0202.01 ◽

2020 ◽

Vol 2 (2) ◽

pp. 1-14

Author(s):

Ammara Akhtar ◽

Sobia Nazir Choudhry ◽

Rana Muhammad Mateen ◽

Mureed Hussain

Keyword(s):

Hereditary Spastic Paraplegia ◽

In Silico ◽

Mitochondrial Protein ◽

In Silico Analysis ◽

Missense Mutations ◽

Spastic Paraplegia ◽

Bioinformatic Tools ◽

Pathogenic Variants ◽

Structural And Functional Properties ◽

Silico Analysis

Hereditary spastic paraplegia (HSP) is a heterogenous neurological disorder primarily associated with progressive spasticity. Paraplegin is a mitochondrial protein and mutations in this protein can lead to HSP. In this study, in silico analysis was carried out to identify the pathogenic variants of SPG7 (paraplegin protein). To find novel pathogenic mutations, missense and splicing variants were collected from gnomAD database and passed through a detailed and stringent analysis with the help of a variety of bioinformatic tools. The list of mutations was examined and compared in ClinVar. Altogether, 14 missense mutations and 18 splicing mutations were obtained and these mutations were predicted to have the potential of disrupting the normal structural and functional properties of paraplegin protein.

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Evolving geographic diversity in SARS-CoV2 and in silico analysis of replicating enzyme 3CLpro targeting repurposed drug candidates

10.21203/rs.3.rs-28084/v2 ◽

2020 ◽

Author(s):

Nitin Chitranshi ◽

Vivek Kumar Gupta ◽

Rashi Rajput ◽

Angela Godinez ◽

Kanishka Pushpitha ◽

...

Keyword(s):

Sequence Alignment ◽

In Silico ◽

Genomic Sequence ◽

In Silico Analysis ◽

Multiple Sequence ◽

Drug Candidates ◽

Recurrent Mutations ◽

Depth Analysis ◽

Repurposed Drugs ◽

Genetic Pool

Abstract Background: Severe acute respiratory syndrome (SARS) has been initiating pandemics since the beginning of the century. In December 2019, the world was hit again by a devastating SARS episode that has so far infected almost four million individuals worldwide, with over 200,000 fatalities having already occurred by mid-April 2020, and the infection rate continues to grow exponentially. SARS coronavirus 2 (SARS-CoV-2) is a single stranded RNA pathogen which is characterised by a high mutation rate. It is vital to explore the mutagenic capability of the viral genome that enables SARS-CoV-2 to rapidly jump from one host immunity to another and adapt to the genetic pool of local populations. Methods: For this study, we analysed 2,301 complete viral sequences reported from SARS-CoV-2 infected patients. SARS-CoV-2 host genomes were collected from The Global Initiative on Sharing All Influenza Data (GISAID) database containing 9 genomes from pangolin-CoV origin and 3 genomes from bat-CoV origin, Wuhan SARS-CoV2 reference genome was collected from GeneBank database. The Multiple sequence alignment tool, Clustal Omega was used for genomic sequence alignment. The viral replicating enzyme, 3-chymotrypsin-like cysteine protease (3CLpro) that plays a key role in its pathogenicity was used to assess its affinity with pharmacological inhibitors and repurposed drugs such as anti-viral flavones, biflavanoids, anti-malarial drugs and vitamin supplements. Results: Our results demonstrate that bat-CoV shares >96% similar identity, while pangolin-CoV shares 85.98% identity with Wuhan SARS-CoV-2 genome. This in-depth analysis has identified 12 novel recurrent mutations in South American and African viral genomes out of which 3 were unique in South America, 4 unique in Africa and 5 were present in-patient isolates from both populations. Using state of the art in silico approaches, this study further investigates the interaction of repurposed drugs with the SARS-CoV-2 3CLpro enzyme, which regulates viral replication machinery. Conclusions: Overall, this study provides insights into the evolving mutations, with implications to understand viral pathogenicity and possible new strategies for repurposing compounds to combat the nCovid-19 pandemic.

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In Silico Characterization of Histidine Acid Phytase Sequences

Enzyme Research ◽

10.1155/2012/845465 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 11

Author(s):

Vinod Kumar ◽

Gopal Singh ◽

A. K. Verma ◽

Sanjeev Agrawal

Keyword(s):

Sequence Alignment ◽

Multiple Sequence Alignment ◽

In Silico ◽

Animal Feed ◽

Protein Sequences ◽

Homology Search ◽

Protein Database ◽

Multiple Sequence ◽

Conserved Sequence ◽

Signature Sequence

Histidine acid phytases (HAPhy) are widely distributed enzymes among bacteria, fungi, plants, and some animal tissues. They have a significant role as an animal feed enzyme and in the solubilization of insoluble phosphates and minerals present in the form of phytic acid complex. A set of 50 reference protein sequences representing HAPhy were retrieved from NCBI protein database and characterized for various biochemical properties, multiple sequence alignment (MSA), homology search, phylogenetic analysis, motifs, and superfamily search. MSA using MEGA5 revealed the presence of conserved sequences at N-terminal “RHGXRXP” and C-terminal “HD.” Phylogenetic tree analysis indicates the presence of three clusters representing different HAPhy, that is, PhyA, PhyB, and AppA. Analysis of 10 commonly distributed motifs in the sequences indicates the presence of signature sequence for each class. Motif 1 “SPFCDLFTHEEWIQYDYLQSLGKYYGYGAGNPLGPAQGIGF” was present in 38 protein sequences representing clusters 1 (PhyA) and 2 (PhyB). Cluster 3 (AppA) contains motif 9 “KKGCPQSGQVAIIADVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDP” as a signature sequence. All sequences belong to histidine acid phosphatase family as resulted from superfamily search. No conserved sequence representing 3- or 6-phytase could be identified using multiple sequence alignment. This in silico analysis might contribute in the classification and future genetic engineering of this most diverse class of phytase.

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Potential of Marine Terpenoids against SARS-CoV-2: An In Silico Drug Development Approach

Biomedicines ◽

10.3390/biomedicines9111505 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1505

Author(s):

Alaka Sahoo ◽

Shivkanya Fuloria ◽

Shasank S. Swain ◽

Sujogya K. Panda ◽

Mahendran Sekar ◽

...

Keyword(s):

Phylogenetic Tree ◽

In Silico ◽

Drug Repurposing ◽

Pharmacokinetic Profile ◽

Genomic Diversity ◽

Ligand Docking ◽

Multiple Sequence ◽

Bioactive Natural Products ◽

Lead Candidate ◽

Development Approach

In an emergency, drug repurposing is the best alternative option against newly emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. However, several bioactive natural products have shown potential against SARS-CoV-2 in recent studies. The present study selected sixty-eight broad-spectrum antiviral marine terpenoids and performed molecular docking against two novel SARS-CoV-2 enzymes (main protease or Mpro or 3CLpro) and RNA-dependent RNA polymerase (RdRp). In addition, the present study analysed the physiochemical-toxicity-pharmacokinetic profile, structural activity relationship, and phylogenetic tree with various computational tools to select the ‘lead’ candidate. The genomic diversity study with multiple sequence analyses and phylogenetic tree confirmed that the newly emerged SARS-CoV-2 strain was up to 96% structurally similar to existing CoV-strains. Furthermore, the anti-SARS-CoV-2 potency based on a protein−ligand docking score (kcal/mol) exposed that the marine terpenoid brevione F (−8.4) and stachyflin (−8.4) exhibited similar activity with the reference antiviral drugs lopinavir (−8.4) and darunavir (−7.5) against the target SARS−CoV−Mpro. Similarly, marine terpenoids such as xiamycin (−9.3), thyrsiferol (−9.2), liouvilloside B (−8.9), liouvilloside A (−8.8), and stachyflin (−8.7) exhibited comparatively higher docking scores than the referral drug remdesivir (−7.4), and favipiravir (−5.7) against the target SARS-CoV-2−RdRp. The above in silico investigations concluded that stachyflin is the most ‘lead’ candidate with the most potential against SARS-CoV-2. Previously, stachyflin also exhibited potential activity against HSV-1 and CoV-A59 within IC50, 0.16–0.82 µM. Therefore, some additional pharmacological studies are needed to develop ‘stachyflin’ as a drug against SARS-CoV-2.

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