Identification and computational studies of kaurene synthase in Stevia rebaudiana MS007 [EMBARGOED]

Abstract

Stevia, a natural sweetener derived from the leaves of the Stevia rebaudiana plant, has emerged as a popular alternative to sugar due to its high sweetness potency and negligible calorie content. The potential health benefits of stevia, including its role in weight management, blood glucose regulation, and its antioxidant properties, were discussed by various researchers. This project reviews the pathways of stevia, focusing on one of the rate-limiting enzymes in the primary pathways to glycosides, kaurene synthase (KS). Kaurene synthase (KS) plays a crucial role in stevia’s terpene biosynthesis pathway, particularly in the production of steviol glycosides (SG), a group of sweetening compounds (e.g., stevioside and rebaudioside). This study outlines the isolation of the KS gene from Stevia Rebaudiana MS007 (stevia MS007) and subsequent in-silico structural examinations, encompassing 3D modeling, molecular docking, and dynamic simulation of the KS gene. The study revealed the functional roles of the sequence region spanning from position 490 to 755 amino acids, inclusive of residue 672. This region acts as both a conserved region and an active site for interactions with metal ions and ligand binding. The KS gene was initially isolated using the conventional Cetyltrimethylammonium bromide (CTAB) method and 2352 bp of the KS nucleic acid sequence was validated through Needleman-Wise pairwise alignment. Through MEGA-X, a phylogenetic tree of KS genes was constructed, uncovering two crucial domains: the DDxxD domain and the NSE/DTE domain, which were identified through INTERPRO. The KS enzyme was categorized as a polar protein with an optimal pH of 5.5 after physicochemical property analysis using PROTPARAM. In the analysis of the three-dimensional structure, the AlphaFold model was chosen as the most promising, based on its highest ERRAT value of 94.3369. Autodock Vina and GROMACS were employed to determine binding interactions and structure-complex stability for the KS enzyme with ent-copalyl diphosphate ligand (ECDP) in 50 ns. During dynamics simulation, the KS enzyme demonstrated exceptional stability, with a low RMSD value of less than 4.0 Å and a minimum distance of 3.0 Å, indicating a robust and stable interaction. The visualization of the protein tunnel provided insight into ligand transport within the protein. The protein tunnel exhibited a length of 9.2 Å with a bottleneck radius of 1.9 Å, indicating optimal conditions for ligand transport. In summary, these findings offer a structural characterization of the stevia MS007 KS protein and establish a foundation for ongoing and future investigations into the KS functional domain.