Amplification of the active site of BnLIP3 gene of Brassica napus L. during germination

Lipases are useful enzymes that are responsible for the hydrolysis of triacylglycerides and play an important role in plant growth. In this study, we report a rapid molecular method to amplify a partial sequence of the lipase class 3 family designated BnLIP3 gene of Brassica napus L. in order to follow its expression and analyze its role during seed germination. Therefore, we conceived PCR homologous primers to amplify the active site encoding region of the BnLIP3 family genes. Subsequently, to sequence determination of the 582 bp fragment, we deduced BnLIP3 specific primers for a nested RTPCR application. The deduced 194 amino acid sequence (Genbank 1160264) was found to share 85% of identity with lipase from Arabidopsis thaliana class 3 family. The GxSxG consensus motif near the catalytic triad at the active serine site was also identified. The peptidic sequence showed little homology with mammalian and microbial lipases. RT-PCR analysis indicated that BnLIP3 gene was expressed during B. napus seed germination.Keywords: Brassica napus L., GxSxG lipase, germination, BnLIP3, RT-PCR.African Journal of Biotechnology Vol. 12(25), pp. 3905-391

Identification of Disalicyloyl Curcumin as a Potential DNA Polymerase Inhibitor for Marek’s Disease Herpesvirus: A Computational Study Using Virtual Screening and Molecular Dynamics Simulations

Marek’s disease virus (MDV) is a highly contagious and persistent virus that causes T-lymphoma in chickens, posing a significant threat to the poultry industry despite the availability of vaccines. The emergence of new virulent strains has further intensified the challenge of designing effective antiviral drugs for MDV. In this study, our main objective was to identify novel antiviral phytochemicals through in silico analysis. We employed Alphafold to construct a three-dimensional (3D) structure of the MDV DNA polymerase, a crucial enzyme involved in viral replication. To ensure the accuracy of the structural model, we validated it using tools available at the SAVES server. Subsequently, a diverse dataset containing thousands of compounds, primarily derived from plant sources, was subjected to molecular docking with the MDV DNA polymerase model, utilizing AutoDock software V 4.2. Through comprehensive analysis of the docking results, we identified Disalicyloyl curcumin as a promising drug candidate that exhibited remarkable binding affinity, with a minimum energy of −12.66 Kcal/mol, specifically targeting the DNA polymerase enzyme. To further assess its potential, we performed molecular dynamics simulations, which confirmed the stability of Disalicyloyl curcumin within the MDV system. Experimental validation of its inhibitory activity in vitro can provide substantial support for its effectiveness. The outcomes of our study hold significant implications for the poultry industry, as the discovery of efficient antiviral phytochemicals against MDV could substantially mitigate the economic losses associated with this devastating disease.Funder: King Saud University, Riyadh, Saudi Arabia; Grant(s): RSPD2023R74

Amplification of the active site of BnLIP3 gene of Brassica napus L. during germination

Lipases are useful enzymes that are responsible for the hydrolysis of triacylglycerides and play an important role in plant growth. In this study, we report a rapid molecular method to amplify a partial sequence of the lipase class 3 family designated BnLIP3 gene of Brassica napus L. in order to follow its expression and analyze its role during seed germination. Therefore, we conceived PCR homologous primers to amplify the active site encoding region of the BnLIP3 family genes. Subsequently, to sequence determination of the 582 bp fragment, we deduced BnLIP3 specific primers for a nested RTPCR application. The deduced 194 amino acid sequence (Genbank 1160264) was found to share 85% of identity with lipase from Arabidopsis thaliana class 3 family. The GxSxG consensus motif near the catalytic triad at the active serine site was also identified. The peptidic sequence showed little homology with mammalian and microbial lipases. RT-PCR analysis indicated that BnLIP3 gene was expressed during B. napus seed germination.Glaied Ghram, I.; Belguith, H.; Messaoudi, A.; Fattouch, S.; Vicente Meana, Ó.; Ben Hamida, J. (2013). Amplification of the active site of BnLIP3 gene of Brassica napus L. during germination. African Journal of Biotechnology. 12(25):3905-3913. doi:10.5897/AJB12.2861S39053913122

Core proteome mediated subtractive approach for the identification of potential therapeutic drug target against the honeybee pathogen Paenibacillus larvae

Background & Objectives American foulbrood (AFB), caused by the highly virulent, spore-forming bacterium Paenibacillus larvae, poses a significant threat to honey bee brood. The widespread use of antibiotics not only fails to effectively combat the disease but also raises concerns regarding honey safety. The current computational study was attempted to identify a novel therapeutic drug target against P. larvae, a causative agent of American foulbrood disease in honey bee. Methods We investigated effective novel drug targets through a comprehensive in silico pan-proteome and hierarchal subtractive sequence analysis. In total, 14 strains of P. larvae genomes were used to identify core genes. Subsequently, the core proteome was systematically narrowed down to a single protein predicted as the potential drug target. Alphafold software was then employed to predict the 3D structure of the potential drug target. Structural docking was carried out between a library of phytochemicals derived from traditional Chinese flora (n > 36,000) and the potential receptor using Autodock tool 1.5.6. Finally, molecular dynamics (MD) simulation study was conducted using GROMACS to assess the stability of the best-docked ligand. Results Proteome mining led to the identification of Ketoacyl-ACP synthase III as a highly promising therapeutic target, making it a prime candidate for inhibitor screening. The subsequent virtual screening and MD simulation analyses further affirmed the selection of ZINC95910054 as a potent inhibitor, with the lowest binding energy. This finding presents significant promise in the battle against P. larvae. Conclusions Computer aided drug design provides a novel approach for managing American foulbrood in honey bee populations, potentially mitigating its detrimental effects on both bee colonies and the honey industry

Three-Dimensional Structure of Lipase Predicted by Homology Modeling Method

Triacylglycerol lipases have been thoroughly characterized in mammals and microorganisms. By contrast, very little is known about plant lipases. In this investigation, a homology model of Arabidopsis thaliana lipase (NP_179126) was constructed using a human gastric lipase (PDB ID: 1HLG), as a template for model building. This model was then assessed for stereochemical quality and side chain environment. Natural substrates: tributyrin, trioctanoin and triolen were docked into the model to investigate ligand-substrate interaction

Classification of EC 3.1.1.3 bacterial true lipases using phylogenetic analysis

Lipases play an important role in lipid metabolism and are produced by a variety of species. All lipases are members of the á/â hydrolase fold super-family. Also, lipases share a conserved active site signature, the Gly-Xaa-Ser-Xaa-Gly motif. To obtain an overview of this industrially and very important class of enzymes and their characteristics, we collected and classified bacterial lipases sequences available from protein databases. Here we proposed an updated and revised classification of family I bacterial "true" lipases based mainly on a comparison of their amino acid sequences and some fundamental physicochemical and biological properties. The result of this work has identified 11 subfamilies of “true” lipases. This work will therefore contribute to a faster identification and to an easier characterization and classification of novel bacterial lipolytic enzymes.Key words: Lipases, phylogenetic analysis, lipolytic enzymes

SUPPLEMENTARY (For MD) An integrative pan-genome and subtractive proteomics approach for the identification of potential novel therapeutic drug target against antibiotic resistant honeybee pathogen Paenibacillus larvae

<p><strong>Parameters</strong></p><p>Force field: AMBER ff19SB</p><p>Water type: TIP3P</p><p>Ions: NaCl  </p><p>Ligand topology force field: GAFF2</p><p>Temperature: 298k</p><p>Pressure: 1 bar</p><p>minimization step:  20000 on  5 nanoseconds</p><p>initial velocity is changed by changing "ntx" and "ig"</p><p>C2: ntx = 5 , ig = 8</p><p>C3: ntx = 2 , ig = 5</p><p> </p><p><strong>Uploads</strong>- </p><p>1. Zip file of all 3 main files</p><p>2. Unzip file of C1 (Trajectory, PDB complex after each 10 ns run, and Mp4 video of Complex)</p><p>3. Zip file of C1</p><p>4. Unzip file of C2 (Trajectory, PDB complex after each 10 ns run, and Mp4 video of Complex)</p><p>5. Zip file of C2</p><p>6. Unzip file of C3 (Trajectory, PDB complex after each 10 ns run, and Mp4 video of Complex)</p><p>7. Zip file of C3</p><p>8. Zip and unzip file of <strong>Initial</strong> PDB of complex prior to MD simulation with <strong>Post</strong> MD PDB (C1, C2, C3)</p><p>9. Zip file of <strong>topology</strong> files for C1, C2, and C3</p&gt