In silico identification and characterization of Kaurene Synthase protein in Stevia rebaudiana MS007

Stevia rebaudiana (Sr), belonging to Asteraceae family is a plant native to Paraguay. It is currently being used as a healthier alternative for sugar. Sr produces steviol glycosides (SGs), a group of secondary metabolite compounds that is responsible for its sweetening taste. SGs act as sweetener due to the presence of two major compounds, Stevioside and Rebaudioside A. Biosynthesis of these compounds involve enzymes such as geranylgeranyl pyrophosphate (GPPS), copalyl diphosphate synthase (CPPS), kaurene synthase (KS) and kaurene oxidase (KO) in the pathway. In this study, the identification and characterization of Stevia rebaudiana MS007 kaurene synthase (SrKS) were done by in silico analysis of the transcriptomic dataset. Homology search from BLASTx resulting in SrKSfrom query Cluster-31069.42907 (Sr MS007) of transcriptomic dataset shows the highest similarity percentage identity (99.62%). ExPasy tools were used to translate the nucleotide sequence into protein sequence. The protein domain is predicted by protein domain search analysis using Interpro and shows IPR005630 (terpene synthase metal-binding domain) available at positions 454 to 719 and IPR001906 (terpene-synthase-N-terminal-domain) at position 222 to 411 as the domains. In constructing the phylogenetic analysis tree, multiple sequence alignment was initially done using MUSCLE and MEGA-X was used as phylogenetic tree analysis tools. Cluster-31069.42907 shows the relationship between the ancestors, based on the bootstrap value. Bootstrap value of Helianthus annuus and Stevia rebaudiana is 100% as both the sequences are from the Asteraceae family. This study contributes to a deeper understanding of S. rebaudiana MS007 Kaurene synthase through in silico analysis

Protein-protein interaction network analysis on the whiteleg shrimp Penaeus vannamei and Vibrio parahaemolyticus host-pathogen relationship reveals possible proteins and pathways involved during infection

Whiteleg shrimp, Penaeus vannamei, is the prime candidate in shrimp aquaculture that significantly contributes to the economic growth of the global aquaculture sector. However, P. vannamei culture is constantly plagued with pathogenic infection of Vibrio spp., including Vibrio parahaemolyticus, which impairs shrimp production and consequently results in significant financial losses. Despite this persistent problem, the relationship between P. vannamei and V. parahaemolyticus remains unclear. The discovery of protein-protein interactions (PPIs) between host and pathogen might improve the understanding of bacterial infection in shrimp since most pathogenic bacteria infect hosts through multiple mechanisms. Hence, this study aims to identify the candidate effector proteins of V. parahaemolyticus and their targeted host proteins and potential pathways involved in its infection using a host-pathogen PPI approach. The P. vannamei – V. parahaemolyticus PPI (PVPPI) network was constructed using in silico methods, followed by clustering and pathway enrichment analyses. The constructed PVPPI network consisted of 448871 interactions between 4427 P. vannamei and 18119 V. parahaemolyticus proteins. Clustering analysis identified several effector proteins, i.e., secretion systems proteins, and their targeted proteins, including cell signalling proteins. The bacterial secretion system, mTOR signalling, and endocytosis were among the enriched pathways that might be involved during V. parahaemolyticus infection in P. vannamei. This study reports the first host-pathogen PPI network between P. vannamei and V. parahaemolyticus, where several hosts and pathogen proteins with potential pathways are highlighted. These findings offer new insights into the interaction between V. parahaemolyticus and P. vannamei, a vital aspect that could serve as a baseline for future disease prevention and treatment in the shrimp aquaculture industry

Complex 1 (ferritin-HrpE/YscL family type III secretion apparatus protein).

The purple chain indicates the ferritin protein from P. vannamei, and the red chain indicates the HrpE/YscL family type III secretion apparatus protein from V. parahaemolyticus. (TIF)</p

Superimposition of pre-MD and post-MD.

The pre-MD for Complex 1, Complex 2, and Complex 3 are represented with yellow, pink, and green, respectively. Meanwhile, the post-MD for Complex 1, Complex 2, and Complex 3, are illustrated with red, purple, and brown, respectively.</p

Workflow of the study.

The rectangle box represents the method. The parallelogram indicates the output.</p

Average of triplicate 100 ns MD simulation.

(a) RMSD, (b) Rg, (c) H-bond, and (d) distance. The purple plot indicates Complex 1 (Ferritin-HrpE/YscL family type III secretion apparatus protein), the red plot represents Complex 2 (Protein kinase domain-containing protein-Chemotaxis CheY protein), and the orange plot illustrates Complex 3 (GPCR-Chemotaxis CheY protein).</p

Protein-protein docking score using the HawkDock server.

Protein-protein docking score using the HawkDock server.</p

Triplicate of 100 ns MD simulation of Complex 2.

(a) RMSD, (b) Rg, (c) Distance, (d) H-bond. The blue plot indicates replicate 1, the red plot represents replicate 2, and the yellow plot illustrates replicate 3.</p

Triplicate of 100 ns MD simulation of Complex 3.

(a) RMSD, (b) Rg, (c) Distance, (d) H-bond. The blue plot indicates replicate 1, the red plot represents replicate 2, and the yellow plot illustrates replicate 3.</p