Abstract
Amyrins are the immediate precursors of many pharmaceutically important pentacyclic triterpenoids. Although various amyrin synthases have been identified, little is known about the relationship between protein structures and the constituent and content of the products. IaAS1 and IaAS2 identified from Ilex asprella in our previous work belong to multifunctional oxidosqualene cyclases and can produce α-amyrin and β-amyrin at different ratios. More than 80% of total production of IaAS1 is α-amyrin; while IaAS2 mainly produces β-amyrin with a yield of 95%. Here, we present a molecular modeling approach to explore the underlying mechanism for selective synthesis. The structures of IaAS1 and IaAS2 were constructed by homology modeling, and were evaluated by Ramachandran Plot and Verify 3D program. The enzyme-product conformations generated by molecular docking indicated that ASP484 residue plays an important role in the catalytic process; and TRP611 residue of IaAS2 had interaction with β-amyrin through π–σ interaction. MM/GBSA binding free energy calculations and free energy decomposition after 50 ns molecular dynamics simulations were performed. The binding affinity between the main product and corresponding enzyme was higher than that of the by-product. Conserved amino acid residues such as TRP257; TYR259; PHE47; TRP534; TRP612; and TYR728 for IaAS1 (TRP257; TYR259; PHE473; TRP533; TRP611; and TYR727 for IaAS2) had strong interactions with both products. GLN450 and LYS372 had negative contribution to binding affinity between α-amyrin or β-amyrin and IaAS1. LYS372 and ARG261 had strong repulsive effects for the binding of α-amyrin with IaAS2. The importance of Lys372 and TRP612 of IaAS1, and Lys372 and TRP611 of IaAS2, for synthesizing amyrins were confirmed by site-directed mutagenesis. The different patterns of residue–product interactions is the cause for the difference in the yields of two products.
Highlights
Oxidosqualene cyclases (OSCs) belong to a multi-gene family of enzymes that convert a linear molecule of 2,3-oxidosqualene (OS) to polycyclic products, such as phytosterols or triterpenoids [1,2,3]
Both α-amyrin and β-amyrin can be synthesized by two multifunctional OSCs, but the yields for the two products differ greatly
A molecular modeling approach by combining homology modeling, structure evaluation, molecular docking, molecular dynamics simulations, Mechanics/Generalized Born Surface Area (MM/GBSA) binding free energy calculations, and free energy decomposition was adopted to explore the mechanism of selective synthesis of amyrins by two oxidosqualene cyclases of Ilex asprella
Summary
Oxidosqualene cyclases (OSCs) belong to a multi-gene family of enzymes that convert a linear molecule of 2,3-oxidosqualene (OS) to polycyclic products, such as phytosterols or triterpenoids [1,2,3]. Triterpenoids are an important class of natural products with wide distribution in animals, fungi, and plants [4,5,6] These structurally diverse triterpenoids have anti-tumor, cognitive enhancement, anti-aging, anti-inflammatory, and hypoglycemic activities [7,8]. Multi-sequence alignment of sequences of IaAS1, IaAS2, human lanosterol synthase and seven enzymes from plants (AsAS, AtCYC, AtLSS1, AtLUP1, EtAS, O2LS, PgAS) provides key information for identifying the interface of enzyme-substrate/intermediate/product interaction (Supplementary Figure S1). Molecular docking and molecular dynamics studies on both enzymes with α-amyrin and β-amyrin could provide more information of their selective catalytic activities. Both thermodynamic and kinetics factors can influence the yields of products. Sci. 2019, 20, 3469 by combining homology modeling, structure evaluation, molecular docking, molecular dynamics simulations, MM/GBSA binding free energy calculations, and free energy decomposition was adopted
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