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.催化混杂性（Catalytic promiscuity）作为酶催化研究领域中一个兼具前沿性却尚未完全明晰的概念，广泛存在于植物来源倍半萜环化酶（sesquiterpene cyclases, FPPC）中，并与倍半萜类天然产物的化学多样性高度相关。烟草来源的5-表-马兜铃烯合酶（Nicotiana tabacum 5-epi-aristolochene synthase, TEAS）与土曲霉来源的马兜铃烯合酶（Aspergillus terreus aristolochene synthase, ATAS）均属于FPPC家族，二者共享相同的反应通路，最终主产物均为马兜铃烯，但二者产物的立体化学构型存在差异。既往实验研究已观测到TEAS的催化混杂性与ATAS的催化保真性，但二者的详细催化机制仍未明确。本研究通过量子力学-经典力学多尺度分子动力学模拟（quantum classical multiscale molecular dynamics simulations）与定点诱变实验（site-directed mutagenesis experiments），系统探究了TEAS中从底物到马兜铃烯及多种副产物的完整酶催化通路，并获得了以下关键机制认知：（1）从底物释放的焦磷酸基团（PPi，diphosphate group）可在TEAS与ATAS的酶催化过程中充当广义酸碱催化剂，该机制大概率在FPPC家族中具有普适性；（2）Asp444-Tyr520二元残基对可作为额外的广义酸碱残基对，提升TEAS的催化混杂性；（3）富集的芳香族残基是维持ATAS催化保真性的核心要素。最后，鉴于不同FPPC的活性口袋均存在不同程度的可塑性，本研究进一步讨论了调控多数FPPC催化混杂性与保真性的三大关键化学控制因素，即底物折叠模式、中间体柔性与关键残基。