Enzyme Promiscuity versus Fidelity in Two Sesquiterpene Cyclases (TEAS versus ATAS)

Catalytic promiscuity, as a modern and imperfect understanding concept in enzyme catalysis community, is prevailing in plant-derived sesquiterpene cyclases (FPPC) and highly related to the chemical diversity of sesquiterpenoid natural products. Both the Nicotiana tabacum 5-epi-aristolochene synthase (TEAS) and Aspergillus terreus aristolochene synthase (ATAS) belong to FPPC, involve the same reaction pathway, and yield their ultimate main product (aristolochene) with different stereochemistries. The catalytic promiscuity of TEAS and fidelity of ATAS have been observed in previous experimental studies, but the detailed catalytic mechanism is still not clear. Herein, by employing the quantum classical multiscale molecular dynamics simulations and site-directed mutagenesis experiments, the complete enzyme catalytic pathways from the substrate to aristolochene and various side products (in TEAS) are investigated and the important mechanism insights are included: (1) the PPi moiety (diphosphate group, released from the substrate) would further act as the general acid/base in both TEAS and ATAS enzyme catalysis, which likely plays a general role in FPPC. (2) The Asp444–Tyr520 dyad acts as an additional general acid/base residue pair to increase promiscuity in TEAS. (3) The enriched aromatic residues are essential for the catalytic fidelity of ATAS. Finally, we further discuss the three critical chemical control factors which are proposed to be responsible for the catalytic promiscuity and fidelity in most FPPC, that is, substrate folding mode, intermediate flexibility, and key residue, owing to the more or less plasticity of the active pocket in various FPPC.