萜烯由异戊二烯焦磷酸衍生而来，组成了一大类化学结构复杂的天然产物。其被广泛应用于医药、化妆品和生物燃料等领域。萜烯化合物最显著的特点是种类多样，目前已知的萜烯约有数万种。萜烯化合物的多样性主要归因于萜烯合成酶。本文将探究萜烯合成酶催化产物多样性的决定因素和酶分子进化机制。 第一章，我们介绍了课题的研究背景和进展。萜烯虽然种类繁多化学结构迥异，但是它们具有相同的底物(geranyl diphosphate GPP、farnesyl diphosphate FPP和geranylgeranyl diphosphate GGPP等)。萜烯合成酶参与异戊二烯焦磷酸环化形成结构各异的萜烯。萜烯合成酶参与的反应被认为是自然界中最复杂的反应。底物或中间体在萜烯合成酶活性口袋经历质子化、异构化、环化、重排和去质子化等反应。单萜烯合成酶参与催化香叶基焦磷酸(GPP)形成最简单萜烯。其中S-柠檬烯合成酶(S-LS)催化合成S-柠檬烯，它是研究单萜烯合成酶催化机理的模式酶。虽然前期对S-LS参与的催化反应进行了大量研究，但其催化产物选择性和酶分子进化机理仍不明确。系统地探究S-LS催化机理和单萜烯合成酶可能的分子进化机制将十分有必要。 第二章，我们通过突变分析和动力学计算讨论了酪氨酸(Y573)在S-LS催化反应中的作用。突变分析表明该酪氨酸的官能团苯环和羟基共同影响催化活性。为了探究Y573可能的作用，对结合芳樟基焦磷酸（LPP）的柠檬烯合成酶晶体结构进行了动力学模拟。模拟结果显示Y573和D496间存在氢键相互作用，以及螺旋型LPP与Y573有紧密的相互作用。为了探究氢键作用对催化反应的影响，我们构建了突变酶D496N。结果显示消除氢键作用的突变酶活性显著下降，这说明该氢键对催化反应非常重要。此外，序列分析表明对应Y573的酪氨酸在合成环形单萜烯的单萜烯合成酶中完全保守而在合成非环形产物的酶中却是可变的。这表明酪氨酸可能参与环化反应或者早期的催化反应。随后使用橙花基二磷酸(NPP)作底物，排除了酪氨酸直接参与或者单独参与异构化的可能。因此酪氨酸在反应中可能起到更加复杂的作用，比如参与控制反应中间体由伸展型向螺旋型构象的转变。 第三章，我们研究了柠檬烯合成酶活性口袋的可塑性以及产物选择性。通过设计突变组合M3(N345A/L423A/S454A)成功将S-LS改造为蒎烯合成酶；或者通过引入较大氨基酸残基构建N345I突变体，使之成为水芹烯合成酶。这表明S-LS活性口袋的残基侧链会影响碳正离子稳定以及其重排路径。进一步研究表明，N345残基的极性对稳定萜品基碳正离子生成柠檬烯起到重要作用。如果其被替换为非极性氨基酸，碳正离子中间体会向焦磷酸基团移动导致发生进一步环化或者氢负离子迁移，产生蒎烯或水芹烯。另一方面，如果突变酶中相同位点仍为极性残基时，催化产物依然是柠檬烯。随后柠檬烯合成酶序列分析表明，对应N345位点残基不完全保守。N345不是唯一参与稳定碳正离子的极性残基，而是多个极性残基组成了一个极性口袋共同发挥稳定碳正离子作用。因此，活性口袋极性决定碳正离子重排进而决定产物选择性。 第四章，我们探究了单萜烯合成酶分子进化机制。S-LS及其部分突变酶的动力学研究结果表明，突变少数氨基酸残基可以显著改变产物专一性。而这些突变酶的催化常数相对野生型酶的无较大变化。这一点可能解释单萜烯的多样性。因为，少数几个氨基酸的突变则可以产生新型的产物，而相对较少变化的催化常数则意味着突变酶所生成的新型萜烯产物会达到一定的浓度，从而使自然选择有可能发生。单萜烯合成酶系统进化树分析揭示了可能的分子进化模型。我们认为被子植物中的柠檬烯合成酶可能由产生较复杂产物的柠檬烯/蒎烯/水芹烯合成酶祖先所进化而来。在进化过程中产生了具有更强极性的活性口袋可能有利于稳定萜品基阳离子，从而提高了柠檬烯合成酶的产物转一性。我们通过在一个蒎烯合成酶活性口袋引入极性氨基酸，成功提高柠檬烯含量，从而验证了该进化模型。 本文为研究单萜烯合成酶催化机理提供了更深入的见解。首先，揭示了酪氨酸(Y573)影响中间体构象变化。其次，发现活性口袋极性决定碳正离子重排和产物选择性。最后，探究了可能的单萜烯合成酶分子进化模型。我们的研究可以解释萜烯多样性，同时可以为定向进化萜烯合成酶合成新型萜烯提供指导。

Terpenes are derived from isoprene pyrophosphate to form one of the largest groups of natural products. They have applications in medicine, cosmetics and biofuel. The most notable characteristics of terpenes are their diversity. It has been estimated that there are tens of thousands of different terpene metabolites. The diversity of terpenes is attributed to terpene synthases. In this study, we investigated the catalytic mechanism and the molecular evolutionary of monoterpene synthases. In the first chapter, we introduced the backgrand of our research. Although terpenes have a wide variety of chemical structures, they are generated from relatively simple substrates, geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), etc. Terpene synthases carry out complex reactions and transform the prenyl diphosphates into skeletal structural diverse terpenoids. In the active site, the substrate or carbocation intermediate undergoes ionization, isomerization, cyclization, rearrangement, deprotonation and other reactions. Monoterpene synthases take part in catalyzing GPP to produce monoterpenes, the simplest terpenes. S-limonene synthase (S-LS) is a model monoterpene synthase that cyclizes GPP to produce S-limonene. A great deal of research has been carried out to investigate the catalytic mechanism of S-LS. However, the products selectivity and the molecular evolutionary mechanism of S-LS are still unknown. Hence, a systematic study to explore the catalytic mechanism and molecular evolution of those enzymes is necessary. In the second chapter, we perform mutagenesis and dynamic simulation to investigate the function of Y573 in LS. Mutational analysis indicates that both the aromatic ring and hydroxyl group are essential for the catalysis. To elucidate the role of Y573, we carried out dynamic simulations on the crystal structures of limonene synthase in complex with linalyl pyrophosphate (LPP). We found a hydrogen bond between Y573 and D496, and also a tight interaction between Y573 and the helical form of the LPP intermediate. Further mutagenesis suggested that this hydrogen bond is essential for catalysis. Furthermore, sequence analysis suggested Y573 is completely conserved among monoterpene synthases that produce cyclic terpene products but variable in enzymes forming acyclic products, indicating this residue may be involved in cyclization or the earlier reactions. Subsequent studies using neryl diphosphate (NPP) as the substrate ruled out the possibility that Y573 functions solely at the substrate isomerization step. Therefore, a more complicated role may be played by this residue. We proposed that Y573 may paly an important role in modifying the conformation of the substrate and intermediates. For example, it may be involved in the transition to the helical form of the LPP intermediate. In the third chapter, we investigated the plasticity and the products selectivity of the active site of S-LS. We are able to convert S-LS to pinene or phellandrene synthases after introducing M3(N345A/L423A/S454A) or N345I mutations. That indicates that the residues near the active site have significantly influence on the product profile. Further studies on N345 suggest the polarity of the residue N345 plays a critical role in limonene production by stabilizing the terpinyl cation intermediate. If it is mutated to a non-polar residue, further cyclization or hydride shifts occurs so the carbocation migrates towards the pyrophosphate, leading to the production of pinene or phellandrene. On the other hand, mutant enzymes that still possess a polar residue at this position produce limonene as the major product. The sequence alignment of limonene synthases suggests that N345 is not the only polar residue that may stabilize the terpinyl cation because it is not strictly conserved among limonene synthases across species. There are also several other polar residues in this area. Therefore, we proposed the existence of a polar pocket in the active site and its polarity may determine the rearrangement of carbocation and the products selectivity. In the fourth chapter, we investigated the molecular evolutionary mechanism of monoterpene synthases. The kinetics features of S-LS and its mutant enzymes were analyzed. Compared with the wild type enzyme, there are only small changes in the catalytic constant of those mutant enzymes despite there are dramatic changes in the product profile. This could expain the chemical diversity of monoterpenes, as new monoterpene products could be easily produced by a few mutations in the monoterpene synthase. And because the catalytic constant does not decrease significantly in the mutant enzyme, there would be a high enough concentration of the new monoterpene, which would allow the nature selection process to take place. By combining with the data from a phylogenetic tree analysis, we proposed a molecular evolutionary model for monoterpene synthases. In this model, angiospermic limonene synthase was evolved from their more promiscuous limonene/pinene/ phellandrene synthases ancestors by introducing polar residues in the active pocket, which increases the product specificity of the enzyme. We were able to increase the yield of limonone by introducing polar residues into the acitive pocket of pinene synthase, which may validate this evolutionary model. Our study provides important insights into the catalytic mechanism of monoterpene synthases. Firstly, it reveals that trysine(Y573) have influence on the conformation of intermediate. Secondly, the polarity of active pocket determines the rearrangement of carbocation and the products selectivity. Finally, we proposed a molecular evolutionary model. This rsearch explains the diversity of terpene. Also, it could provide the guidance for the directed evolution of monoterpene synthses to produce novel products.