Molecular Dynamics Simulations Elucidate Conformational Dynamics Responsible for the Cyclization Reaction in TEAS

Abstract

The Mg-dependent 5-epi-aristolochene synthase from Nicotiana tabacum (called TEAS) could catalyze the linear farnesyl pyrophosphate (FPP) substrate to form bicyclic hydrocarbon 5-epi-aristolochene. The cyclization reaction mechanism of TEAS was proposed based on static crystal structures and quantum chemistry calculations in a few previous studies, but substrate FPP binding kinetics and protein conformational dynamics responsible for the enzymatic catalysis are still unclear. Herein, by elaborative and extensive molecular dynamics simulations, the loop conformation change and several crucial residues promoting the cyclization reaction in TEAS are elucidated. It is found that the unusual noncatalytic NH2-terminal domain is essential to stabilize Helix-K and the adjoining J-K loop of the catalytic COOH-terminal domain. It is also illuminated that the induce-fit J-K/A-C loop dynamics is triggered by Y527 and the optimum substrate binding mode in a "U-shape" conformation. The U-shaped ligand binding pose is maintained well with the cooperative interaction of the three Mg(2+)-containing coordination shell and conserved residue W273. Furthermore, the conserved Arg residue pair R264/R266 and aromatic residue pair Y527/W273, whose spatial orientations are also crucial to promote the closure of the active site to a hydrophobic pocket, as well as to form π-stacking interactions with the ligand, would facilitate the carbocation migration and electrophilic attack involving the catalytic reaction. Our investigation more convincingly proves the greater roles of the protein local conformational dynamics than do hints from the static crystal structure observations. Thus, these findings can act as a guide to new protein engineering strategies on diversifying the sesquiterpene products for drug discovery.

Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

“…In amorphadiene synthase, the aromatic phenylalanine residue (residue in the same position of Y523 of Pam Tps1) was similarly involved in positioning of the FPP substrate in the active site, which subsequently stabilized the carbocation intermediates 84 . A similar observation was also reported by Zhang et al 85 with Nicotiana tabacum 5-epi-aristolochene synthase (TEAS) that catalyzed the cyclisation of FPP into bicyclic 5- epi -aristolochene. Mutational analysis of the aromatic amino acids proved the essential role of these residues in the active site for stabilization of the carbocation intermediates 79 , 85 .…”

“…A similar observation was also reported by Zhang et al 85 with Nicotiana tabacum 5-epi-aristolochene synthase (TEAS) that catalyzed the cyclisation of FPP into bicyclic 5- epi -aristolochene. Mutational analysis of the aromatic amino acids proved the essential role of these residues in the active site for stabilization of the carbocation intermediates 79 , 85 . Positioning of GPP and FPP in the Pam Tps1 active site surrounded by these aromatic residues suggested that this docking analysis was rational and compatible with other crystal structures of terpene synthases.…”

In planta and in silico characterization of five sesquiterpene synthases from Vitis vinifera (cv. Shiraz) berries

“…Residues outside the active site are thus likely to play an important role here. For instance, residues in helix 4 of the N-terminal domain appeared important for the functionality of some sesquiterpene synthases from Aquilaria crassna (Kumeta and Ito 2010) and a molecular dynamic study of TEAS points to the N-terminal domain in facilitating the stability of helix K and the ordering of the J-K loop (Zhang et al 2016). However, characterization in planta or using microorganisms may also be a contributing factor to this observed difference in germacrene D production (Salvagnin et al 2016) and the heat in the GC-MS may also have an impact (Andersen et al 2015).…”

Biosynthetic potential of sesquiterpene synthases: product profiles of Egyptian Henbane premnaspirodiene synthase and related mutants

“…A recent computational analysis of the TEAS active site confirms the importance of this 'pre-Germacrene' conformation of FPP. 31 The 'U-shape' of FPP observed in the active site of our crystal structure is induced by the coordination of the pyrophosphate by the three Mg 2+ ions. The U shape is important for the conformation of the J-K loop, and more specifically, proper orientation of Y527.…”